Wu Huijuan, Wang Suxia, Xue Aimin, Liu Yuan, Liu Ye, Wang Huijun, Chen Qi, Guo Muyi, Zhang Zhigang
Department of Pathology and Key Laboratory of Molecular Medicine, Ministry of Education of China, Fudan University, Shanghai, China.
Nephrology (Carlton). 2008 Oct;13(7):607-15. doi: 10.1111/j.1440-1797.2008.00961.x. Epub 2008 Jun 1.
Decorin (DCN) is a small leucine-rich proteoglycan that plays an important role in the regulation of intercellular contact, cell migration and proliferation. DCN suppresses cell growth and induces apoptosis in various tumour cells. The aim of this study was to investigate whether overexpression of DCN could induce apoptosis and cell growth arrest in mesangial cells (MsCs) in vitro.
PcDNA3.1A-DCN plasmid was transfected into cultured rat MsCs, and positive clones stably expressing DCN (MsC/DCN) were selected. SiRNA was used for blocking DCN expression in MsC/DCN. Apoptosis and cell growth of MsCs were assayed by flow cytometry. Hoechst staining was used for observing apoptotic cells. Expressions of active Caspase-3, epidermal growth factor receptor (EGFR), P21 and transforming growth factor-beta (TGF-beta1) were analyzed using Western blot.
Overexpression of DCN in MsCs induced apoptosis and arrested cells in the G(0)/G(1) phase. The protein level of active Caspase-3 was significantly elevated in MsC/DCN (P < 0.01). DCN transfection induced downregulation of EGFR and up-expression of P21. In addition, the expression of TGF-beta1 was significantly inhibited. DCN-siRNA transfection remarkably blocked the expression of DCN and reversed the downregulatory effects of DCN on MsC's proliferation.
Overexpression of DCN could inhibit MsCs proliferation by inducing apoptosis and cell growth arrest in vitro and it also downregulates expression of TGF-beta1. These results suggest novel strategies for regulating the proliferation of MsC in glomerular diseases.
核心蛋白聚糖（DCN）是一种富含亮氨酸的小分子蛋白聚糖，在调节细胞间接触、细胞迁移和增殖中起重要作用。DCN可抑制多种肿瘤细胞的生长并诱导其凋亡。本研究旨在探讨DCN过表达是否能在体外诱导系膜细胞（MsCs）凋亡和细胞生长停滞。
将PcDNA3.1A-DCN质粒转染至培养的大鼠MsCs中，筛选出稳定表达DCN的阳性克隆（MsC/DCN）。使用小干扰RNA（SiRNA）阻断MsC/DCN中DCN的表达。通过流式细胞术检测MsCs的凋亡和细胞生长情况。采用Hoechst染色观察凋亡细胞。使用蛋白质免疫印迹法分析活化的半胱天冬酶-3、表皮生长因子受体（EGFR）、P21和转化生长因子-β（TGF-β1）的表达。
MsCs中DCN过表达诱导细胞凋亡并使细胞停滞于G(0)/G(1)期。MsC/DCN中活化的半胱天冬酶-3蛋白水平显著升高（P < 0.01）。DCN转染导致EGFR下调和P21上调。此外，TGF-β1的表达受到显著抑制。DCN-siRNA转染显著阻断了DCN的表达，并逆转了DCN对MsC增殖的下调作用。
DCN过表达可通过在体外诱导凋亡和细胞生长停滞来抑制MsCs增殖，并且还下调TGF-β1的表达。这些结果提示了调节肾小球疾病中MsC增殖的新策略。
