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H-RasV12诱导的人成纤维细胞早期衰老中自噬的调节
王玲1△,赵丹3△,余乐1,李亦蕾2*
0
(1. 南方医科大学药学院, 广州 510515;
2. 南方医科大学南方医院药学部, 广州 510515;
3. 解放军第85医院药剂科, 上海 200052
共同第一作者
*通信作者)
摘要:
目的 研究癌基因H-RasV12过度表达或激活与自噬活性的关系,在人成纤维细胞中观察Ras过度表达对自噬活性的影响。方法 在BJ人成纤维细胞中转染原癌基因H-RasV12或其空载体,通过形态学、细胞生长曲线、β-半乳糖苷染色、衰老和自噬相关蛋白表达、流式细胞分析、siRNA(small interfering RNA,siRNA)抑制自噬关键基因5(ATG5),来分析Ras过度表达的细胞效应。结果 与对照细胞相比,H-RasV12过度表达的BJ细胞出现明显的早期衰老,其自噬活性受到抑制,表现为蛋白p62和微管相关蛋白1轻链3Ⅱ( Light chain 3Ⅱ,LC3Ⅱ)的显著蓄积,在研究时间区间内这种抑制作用保持稳定,同时细胞凋亡率增加;利用siRNA抑制ATG5表达,抑制自噬可进一步加重衰老。结论 人成纤维细胞稳定表达癌基因H-RasV12后,可出现自噬活性受到抑制的现象,且自噬抑制处于自噬过程的后期。这一现象可能与Ras相关性癌症的癌变机制有关。
关键词:  自噬  H-RasV12  过早衰老  成纤维细胞
DOI:10.3724/SP.J.1008.2014.01191
投稿时间:2014-06-04修订日期:2014-10-19
基金项目:国家自然科学基金(81102475);南方医科大学南方医院高层次课题匹配基金(81102475).
Autophagy activity is inhibited in human fibroblast cells stably overexpressing H-RasV12
WANG Ling1△,ZHAO Dan3△,YU Le1,LI Yi-lei2*
(1. School of Pharmaceutical Science, Southern Medical University, Guangzhou 510515, Guangdong, China;
2. Department of Pharmacology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong, China;
3. Department of Pharmacology, No.85 Hospital of PLA, Shanghai 200052, China
Co-first authors.
*Corresponding authors)
Abstract:
Objective To investigate the relationship between oncogenic H-RasV12 overexpression/activation and the autophagic activity by observing the effect of Ras overexpression on autophagic activity in human fibroblast cells. Methods Human BJ fibroblast cells were transfected with H-RasV12 or control vector, and then the cellular responses to H-RasV12 overexpression were analyzed by observing the morphology, cell growth curve, senescence-associated β-Gal staining, Western blotting analysis, flow cytometry, and suppression of autophagy-related protein 5 (ATG5) by siRNA. Results Compared with control group, BJ cells overexpressing H-RasV12 developed prominent premature senescence and inhibited autophagic activity, as manifested by significant accumulation of p62 and light chain 3Ⅱ (LC3Ⅱ). The autophagy inhibition by H-RasV12 remained stable during the study period; the apoptosis rate was increased in H-RasV12 overexpressing BJ cells compared with that in the control cells. Suppression of ATG5 by siRNA led to more severe senescence in Ras-overexpressing BJ cells. Conclusion Our results suggest that the autophagy activity is inhibited in human fibroblast cells stably overexpressing oncogenic H-RasV12, and the inhibition is in the later stage of autophagy, which may be related to H-RasV12-related tumorigenesis.
Key words:  autophagy  H-RasV12  premature aging  fibroblasts