Beclin-1 基因沉默对前列腺增生上皮细胞自噬和凋亡的影响。

Effect of Beclin-1 gene silencing on autophagy and apoptosis of the prostatic hyperplasia epithelial cells.

机构信息

Department of Urology, Yangpu Hospital, School of Medicine, Tongji University, Shanghai, China; Department of Urology, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian, China.

Department of Urology, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian, China.

出版信息

Clinics (Sao Paulo). 2022 Sep 8;77:100076. doi: 10.1016/j.clinsp.2022.100076. eCollection 2022.

DOI:10.1016/j.clinsp.2022.100076

PMID:36088885

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9468350/

Abstract

OBJECTIVES

This study aims to explore the effect of silencing Beclin-1 gene on autophagy and apoptosis of Benign Prostatic Hyperplasia (BPH) (BPH-1) cells under the condition of Androgen Deprivation (AD) and Autophagy Inhibition (AI).

METHODS

Control group (BPH-1 group), empty carrier group (sh-RNA-BPH-1 group) and Beclin-1 silenced group (sh-Beclin1-BPH-1 group) were set. The Beclin-1 gene silencing efficiency was detected by RT-PCR and Western blot. Autophagic flux was monitored by GFP-LC3 cleavage assay and cell apoptosis was analyzed by flow cytometry. The protein expression levels of LC3, Caspase-3, PARP-1, Bcl-2, and Bax were detected by Western blot.

RESULTS

The transfection of sh-Beclin-1 obviously down-regulated the expression of Beclin-1 at both mRNA and protein levels. Under the conditions of AD and AI, silencing of Beclin-1 restrained the autophagy of BPH-1 cells, as evidenced by a decreased number of autophagosomes and down-regulation of LC3-II protein (p < 0.001). The results of flow cytometry showed that the apoptotic rate of sh-Beclin-1 group was elevated significantly compared to the other two groups (p < 0.01). Western blot results showed that silencing of Beclin-1 promoted 89 kd fragmentation of PARP-1 (p < 0.001) and Caspase-3 activation (p < 0.01). Moreover, silencing of Beclin-1 resulted in declined Bcl-2 and augmented Bax protein expression in BPH-1 cells (p < 0.01), which ultimately led to a decreased Bcl-2/Bax ratio.

CONCLUSIONS

The results indicated that the silencing of Beclin-1 gene hampered autophagy while activating apoptosis in BPH-1 cells. Thus, Beclin-1 may participate in an antagonistic relationship between autophagy and apoptosis in BPH.

摘要

目的

本研究旨在探讨沉默 Beclin-1 基因对雄激素剥夺（AD）和自噬抑制（AI）条件下良性前列腺增生（BPH）（BPH-1）细胞自噬和凋亡的影响。

方法

设置对照组（BPH-1 组）、空载组（sh-RNA-BPH-1 组）和 Beclin-1 沉默组（sh-Beclin1-BPH-1 组）。通过 RT-PCR 和 Western blot 检测 Beclin-1 基因沉默效率。通过 GFP-LC3 切割试验监测自噬流，通过流式细胞术分析细胞凋亡。Western blot 检测 LC3、Caspase-3、PARP-1、Bcl-2 和 Bax 的蛋白表达水平。

结果

sh-Beclin-1 的转染明显下调了 Beclin-1 在 mRNA 和蛋白水平的表达。在 AD 和 AI 条件下，沉默 Beclin-1 抑制了 BPH-1 细胞的自噬，表现为自噬体数量减少和 LC3-II 蛋白下调（p<0.001）。流式细胞术结果显示，与其他两组相比，sh-Beclin-1 组的凋亡率显著升高（p<0.01）。Western blot 结果表明，沉默 Beclin-1 促进了 PARP-1（p<0.001）和 Caspase-3 激活（p<0.01）的 89kd 片段化。此外，沉默 Beclin-1 导致 BPH-1 细胞中 Bcl-2 表达下调和 Bax 蛋白表达增加（p<0.01），最终导致 Bcl-2/Bax 比值降低。