Yang Chuang, Han Li-Ou
Third Department of General Surgery, the Second Affiliated Hospital of Harbin Medical University, Harbin 150086, P.R. China.
Mol Med Rep. 2010 Sep-Oct;3(5):801-7. doi: 10.3892/mmr.2010.347. Epub 2010 Jul 26.
The aim of the present study was to investigate the effects of Beclin 1 knockdown on spontaneous and vitamin K3 (VK3)-regulated autophagy, survival and apoptosis in human hepatocarcinoma SMMC-7721 cells, and to explore the potential mechanisms underlying the action of Beclin 1 knockdown in the processes of autophagy and apoptosis. A recombinant plasmid-expressing small interfering RNA (siRNA) targeting Beclin 1 mRNA was constructed and introduced into SMMC-7721 cells. The expression of Beclin 1 was determined by reverse transcription-polymerase chain reaction and Western blotting. Subsequently, the impact of Beclin 1 knockdown on spontaneous and VK3-induced autophagy, survival and apoptosis was determined. The expression of cyclin D1, cyclin-dependent kinase 4 (CDK4), Bcl-2, Bcl-xL and the activation of caspase-3 were examined by Western blotting. Transfection with the plasmid for Beclin 1 siRNA expression dramatically down-regulated Beclin 1 expression in SMMC-7721 cells. The knockdown of Beclin 1 expression significantly inhibited spontaneous and VK3-induced autophagy, but did not affect spontaneous proliferation and apoptosis in SMMC-7721 cells in vitro. By contrast, the silencing of Beclin 1 expression significantly enhanced the inhibition of survival and proliferation by VK3, and promoted VK3-induced apoptosis by significantly down-regulating cyclin D1, CDK4, Bcl-2 and Bcl-xL expression and enhancing caspase-3 activation in SMMC-7721 cells in vitro. Our data indicate that Beclin 1 is a positive regulator of autophagy, but a negative regulator of VK3-induced apoptosis in human hepatoma cells.
本研究旨在探讨Beclin 1基因敲低对人肝癌SMMC-7721细胞自噬、存活及凋亡的影响，以及维生素K3（VK3）对上述过程的调控作用，并探究Beclin 1基因敲低在自噬和凋亡过程中发挥作用的潜在机制。构建了靶向Beclin 1 mRNA的重组表达小干扰RNA（siRNA）质粒，并将其导入SMMC-7721细胞。通过逆转录聚合酶链反应和蛋白质免疫印迹法检测Beclin 1的表达。随后，确定Beclin 1基因敲低对自噬、存活及凋亡的影响。通过蛋白质免疫印迹法检测细胞周期蛋白D1、细胞周期蛋白依赖性激酶4（CDK4）、Bcl-2、Bcl-xL的表达及半胱天冬酶-3的激活情况。转染Beclin 1 siRNA表达质粒后，SMMC-7721细胞中Beclin 1的表达显著下调。Beclin 1表达的敲低显著抑制了自噬及VK3诱导的自噬，但对SMMC-7721细胞的自发增殖和凋亡无影响。相反，Beclin 1表达的沉默显著增强了VK3对细胞存活和增殖的抑制作用，并通过显著下调细胞周期蛋白D1、CDK4、Bcl-2和Bcl-xL的表达及增强半胱天冬酶-3的激活促进了VK3诱导的凋亡。我们的数据表明，Beclin 1是自噬的正调控因子，但却是人肝癌细胞中VK3诱导凋亡的负调控因子。
