Beclin1在MG132抗OVCAR3卵巢癌细胞中的变化及作用研究

The change and effect of Beclin 1 on cytotoxicity of ovarian cancer cells induced by MG132

目的:研究Beclin 1在MG132抗OVCAR3卵巢癌细胞中的变化及作用。方法:OVCAR3细胞经不同浓度MG132处理24h后,MTT法检测细胞存活率,Western blot法检测自噬相关Beclin 1蛋白的变化。采用细胞转染技术过表达卵巢癌细胞中Beclin 1,MG132处理24h后,MTT法检测细胞存活率;Hoechst 33258进行核染色观察染色质浓缩和核碎片等凋亡特征性形态学改变。采用shRNA技术下调OVCAR3细胞的Beclin 1表达,MG132处理细胞,AO染色观察酸性自噬泡的形成。结果:与空白对照组比较,1、2、5和10μmol/L浓度的MG132作用24h均能明显抑制OVCAR3细胞的生长(均P<0.05),5μmol/L浓度接近半抑制率;而且5μmol/L和10μmol/L浓度的MG132作用24h能显著诱导OVCAR3细胞的Beclin 1蛋白水平下降(P=0.001)。与空质粒转染组对比,过表达Beclin 1的卵巢癌细胞在MG132处理后核的凋亡明显增加;细胞存活率明显降低,(P=0.00089)。下调卵巢癌细胞中Beclin 1表达后,MG132诱导的酸性囊泡蓄积无改变。结论:MG132使OVCAR3细胞中Beclin 1降低,Beclin 1具有增强MG132抗OVCAR3的作用,但该作用与自噬无关。

Objective：To investigate the change and the effect of Beclin 1 on cytotoxicity of ovarian cancer ceils mediated by proteasome inhibitor MG132. nethods：OVCAR3 cells were treated with indicated concentrations of proteasome inhibitor MG132 for 24h,cell viability was measured using MTT assay and beclin 1 expression was detected using Western blot. OVCAR3 cells were transfected with mock or Beclin 1 eukaryotic plasmid, then treated with MG132 ,cell viability was measured using MTT assay; Nuclear morphology was analyzed using Hoechst 33258 staining. OVCAR3 cells were transfected with Beclin 1 specific shRNA（ shBeclin 1 ） ,then treated with MG132 for 24h, the formation of acidic vacuoles were analyzed using AO staining. Results： 1,2,5,10 μ mol/L MG132 resulted in suppression of cell growth（ P 〈 0.05） ;SbLmol/L and 10μmol/L MG132 reduced Beclin ! expression at the protein levels in OVCAR3 cells（ both P = 0. 001 ）. Overexpression of Beclin 1 enhanced the cytotoxicity of OVCAR3 cells mediated by MGI32（P = 0. 00089）, and apoptotic morphological characteristics turned more predominant. Downregulation of Beclin 1 by shRNA demonstrated no obvious effect on accumulation of acidic vesicular organelles induced by MGI32. Conclusion：MG132 reduced the Beclin 1 level in OVCAR3 cells, Beclin 1 enhanced the cytotoxicity of OVCAR3 cells mediated by MG132 in an autophagy- independent manner.