Silencing of MST1 expression by siRNA diminishes TNF-α- mediated human umbilical vein endothelial cell apoptosis.
10.3969/j.issn.1672-7347.2012.07.004
- Author:
Wei RUAN
1
;
Suobei LI
;
Junmei XU
;
Feng XIAO
;
Rong TAN
Author Information
1. Department of Anesthesiology, Central South University, Changsha, China.
- Publication Type:Journal Article
- MeSH:
Apoptosis;
genetics;
Cells, Cultured;
Hepatocyte Growth Factor;
genetics;
metabolism;
Human Umbilical Vein Endothelial Cells;
cytology;
Humans;
Proto-Oncogene Proteins;
genetics;
metabolism;
RNA Interference;
RNA, Messenger;
genetics;
metabolism;
RNA, Small Interfering;
genetics;
Tumor Necrosis Factor-alpha;
pharmacology
- From:
Journal of Central South University(Medical Sciences)
2012;37(7):669-674
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To elucidate the effects of mammalian sterile 20-like kinase 1 (MST1) gene on tumor necrosis factor (TNF)-α-mediated human umbilical vein endothelial cell (HUVEC) apoptosis.
METHODS:Cultured HUVECs were treated with either vehicle or TNF-α (1-100 ng/mL) for 24 hours. Cell apoptosis was measured by TUNEL staining, and MST1 activity was analyzed by Western blot. In order to knock down MST1 expression in HUVECs, cells were transfected with 100 nmol/L MST1 small interference RNA (siRNA) using Lipofectamine 2000 for 24 hours, and the transfection efficiency was analyzed by Western blot. MST1 siRNA-transfected cells were treated with 10 ng/mL TNF-α for an additional 24 hours. Cell apoptosis was measured by TUNEL staining and caspase-3 activity was detected by Western blot.
RESULTS:MST1 activity was stimulated in a dose-dependent manner after TNF-α treatment (10, 40, 100 ng/mL) and reached the maximal effect at 100 ng/mL. MST1 activity also paralleled the onset of apoptosis as determined by TUNEL staining (P<0.001). Transfection with MST1 siRNA markedly diminished MST1 gene expression in a dose-dependent manner. MST1 siRNA (100 nmol/L) significantly silenced MST1 gene (P<0.05) and reduced TNF-α-induced endothelial cells apoptosis (P<0.05) by way of inhibiting MST1 gene activation and, accordingly, suppressing caspase-3 activity.
CONCLUSION:Silencing of MST1 expression by siRNA diminishes TNF-α-mediated human umbilical vein endothelial cell apoptosis by inhibiting the cascade effect of caspase-3.
