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

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

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、BAX、Bcl2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、H_2O_2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H_2O_2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、BAX、Bcl2、LC3B、p-AKT、m TOR等表达量下降。结论 Beclin 1在裸鼹鼠成纤维细胞抵抗饥饿、H_2O_2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。

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 H_2O_2 treatment. 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 1 gene at mRNA and protein levels was significantly lower in fibroblasts of the naked mole rat. Starvation and H_2O_2 treatment 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,LC3 B,p-AKT and m TOR 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 H_2O_2 stimulation. Inhi-bition 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.