RNA干扰抑制Beclin1基因对裸鼹鼠成纤维细胞增殖与凋亡的影响
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国家科技支撑计划(No.2015BAI09B02);国家自然科学基金(No.31402028)与上海市卫计委课题(No.20144Y0205)联合资助。


Inhibitory effects of Beclin 1gene expression by RNA interference on the proliferation and apoptosis in fibroblasts of naked mole rat
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    摘要:

    目的应用RNA干扰技术抑制自噬调控基因Beclin 1的表达,检测Beclin 1表达对裸鼹鼠皮肤成纤维细胞增殖与凋亡的影响以及p53、BAXBcl2等基因表达的影响。方法分别检测裸鼹鼠成纤维细胞经饥饿、H2O2刺激等处理后Beclin 1的表达,然后采用设计的Beclin l基因的干扰RNA及阴性对照分别瞬时转染裸鼹鼠成纤维细胞。采用real-time PCR及Western blot法检测沉默效果后,采用CCK-1实验检测沉默后细胞增殖活性,流式细胞仪检测细胞凋亡情况,然后采用Western blot检测相关基因蛋白表达水平。结果饥饿与H2O2刺激均能导致Beclin 1表达水平的改变。采用gene expresso转染试剂对裸鼹鼠皮肤成纤维细胞转染效率可达到90%以上,real-time PCR及Western blot结果显示所设计的Beclin 1 siRNA可有效降低Beclin 1的表达。Beclin 1基因沉默后,裸鼹鼠皮肤成纤维细胞增殖抑制率均显著高于对照组,细胞早期凋亡与晚期凋亡率均显著升高,同时p53、BAXBcl2、LC3Bp-AKTmTOR等表达量下降。结论Beclin 1在裸鼹鼠成纤维细胞抵抗饥饿、H2O2刺激等过程中表达量显著变化,同时抑制Beclin 1的表达,可抑制裸鼹鼠细胞增殖,促进其凋亡,这提示Beclin 1基因对裸鼹鼠自噬、增殖、凋亡起到调控作用。

    Abstract:

    Objective To investigate the effects of down-regulation of Beclin 1, which is an autophagy regulatory molecule, expression induced by RNA interference on the proliferation and apoptosis in skin fibroblasts of naked mole rat. Methods The expression levels of Beclin 1 were detected after starvation or H2O2treatment. The fibroblasts were transiently transfected with specific siRNA targeting Beclin 1 and then screened by real-time PCR and Western blot. Cell proliferation and apoptosis were determined using CCK-8 detection kit and flow cytometry (FCM). The expressions of related genes were detected by Western blot. Results The expression of Beclin 1gene at mRNA and protein levels was significantly lower in fibroblasts of the naked mole rat. Starvation and H2O2treatment induced changes of the Beclin 1 expression. Inhibition of Beclin 1 gene expression can inhibit cell proliferation and induce early and late apoptosis. The protein levels of p53, BAX, Bcl2, LC3B, p-AKT and mTOR were reduced after transient transfection with Beclin 1-siRNA. Conclusions The expression of Beclin 1 in fibroblasts of naked mole rat are changed in response to starvation or H2O2 stimulation. Inhibition of Beclin 1 gene expression can inhibit cell proliferation and induce apoptosis. Therefore, Beclin1 gene may play a regulatory role in autophagy, proliferation and apoptosis in the skin fibroblasts of naked mole rat.

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赵善民,肖邦,林丽芳,宫晨,王运慧,程继帅,余琛琳,丛薇,汤球,孙伟,崔淑芳. RNA干扰抑制Beclin1基因对裸鼹鼠成纤维细胞增殖与凋亡的影响[J].中国实验动物学报,2015,23(6):557~561,566.

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  • 收稿日期:2015-11-15
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  • 在线发布日期: 2015-12-30
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