Related Experiment Video
Updated: May 30, 2026
A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9
Published on: August 28, 2018
PCSK9 siRNA inhibits HUVEC apoptosis induced by ox-LDL via Bcl/Bax-caspase9-caspase3 pathway
Chun-Yan Wu1, Zhi-Han Tang, Lu Jiang
1Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, University of South China, Hengyang, 421001, Hunan, China.
Abstract:
This paper investigated the effects of ox-LDL on PCSK9, and the molecular mechanisms of PCSK9 siRNA-inhibited apoptosis induced by ox-LDL in human umbilical vein endothelial cells (HUVECs), to clarify the role of PCSK9 in atherosclerogenesis. HUVECs were incubated with ox-LDL for 24 h. The apoptosis was observed by Hoechst 33258 staining. The expression of PCSK9, LOX-1 mRNAs and proteins was detected by RT-PCR, western blot, respectively. The PCSK9 siRNAs labeled with fluorescence were transfected into HUVECs by Lipofectamine 2000. After transfection for 24 h, cells were treated with ox-LDL for 24 h, HUVECs apoptosis transfected siRNA was detected by Hoechst 33258 staining and flow cytometer. The expression of Bcl-2, Bax, caspase3, 8, 9 was detected by western blot. The activity of caspase3, 9 was detected by kits. Our results showed that apoptosis of HUVECs and the expressions of PCSK9 and LOX-1 were upregulated secondary to induction by ox-LDL in a concentration-dependent manner. However, ox-LDL-induced HUVEC apoptosis and PCSK9 expression, but not LOX-1 expression, were significantly reduced by PCSK9 siRNA. These results demonstrate a linkage between HUVEC apoptosis and PCSK9 expression. Furthermore, we detected the possible pathway involved in apoptotic regulation by PCSK9 siRNA; our results showed that the expression of Bcl-2 decreased, whereas that of Bax increased. In addition, ox-LDL enhanced the activity of caspase9 and then caspase3. Pretreatment of HUVECs with PCSK9 siRNA blocked these effects of ox-LDL. These findings suggest that ox-LDL-induced HUVECs apoptosis could be inhibited by PCSK9 siRNA, in which Bcl/Bax-caspase9-caspase3 pathway maybe was involved through reducing the Bcl-2/Bax ratio and inhibited the activation of both caspase9 and 3.
Insights
Oxidized low-density lipoprotein (ox-LDL) increases apoptosis in human umbilical vein endothelial cells (HUVECs) by upregulating PCSK9. PCSK9 siRNA inhibits this ox-LDL-induced apoptosis, revealing PCSK9
Area of Science:
- Molecular Biology
- Cardiovascular Research
- Cell Biology
Background:
- Oxidized low-density lipoprotein (ox-LDL) plays a critical role in the pathogenesis of atherosclerosis.
- Proprotein convertase subtilisin/kexin type 9 (PCSK9) is implicated in lipid metabolism and cardiovascular disease.
- The specific role of PCSK9 in ox-LDL-induced endothelial cell apoptosis requires further elucidation.
Purpose of the Study:
- To investigate the effect of ox-LDL on PCSK9 expression in human umbilical vein endothelial cells (HUVECs).
- To explore the molecular mechanisms by which PCSK9 mediates ox-LDL-induced apoptosis in HUVECs.
- To clarify the role of PCSK9 in atherosclerogenesis.
Main Methods:
- HUVECs were treated with varying concentrations of ox-LDL.
- Apoptosis was assessed using Hoechst 33258 staining and flow cytometry.
- PCSK9 and LOX-1 expression (mRNA and protein) were measured by RT-PCR and Western blot.
- PCSK9 siRNA was used to inhibit PCSK9 expression, followed by ox-LDL treatment.
- Expression of apoptosis-related proteins (Bcl-2, Bax, caspases) and caspase activity were analyzed.
Main Results:
- Ox-LDL upregulated HUVEC apoptosis, PCSK9, and LOX-1 expression in a concentration-dependent manner.
- PCSK9 siRNA significantly reduced ox-LDL-induced HUVEC apoptosis and PCSK9 expression, but not LOX-1 expression.
- PCSK9 siRNA treatment reversed the ox-LDL-induced decrease in Bcl-2 and increase in Bax expression.
- Ox-LDL-induced activation of caspase-9 and caspase-3 was inhibited by PCSK9 siRNA.
Conclusions:
- PCSK9 plays a significant role in mediating ox-LDL-induced apoptosis in HUVECs.
- PCSK9 siRNA demonstrates a protective effect against ox-LDL-induced endothelial cell apoptosis.
- The Bcl-2/Bax-caspase-9-caspase-3 pathway is involved in PCSK9-mediated apoptosis regulation.