摘要
目的:在研究内质网应激介导的细胞凋亡过程中,我们发现Ring finger protein13(RNF13)具有促进细胞凋亡的功能。我们拟研究沉默RNF13后细胞对Tunicamycin等引起的细胞凋亡的影响,以及RNF13对活性形式的caspase3,XBP1(X-box binding protein 1)的剪切以及IRE1(Endoplasmic reticulum to nucleus signaling 1)磷酸化的影响以有助于了解RNF13促进细胞凋亡的信号通路的研究。方法:基因沉默RNF13,利用MTT方法研究RNF13沉默后对细胞增殖的影响,RNF13基因沉默后对XBP1剪切的影响,免疫印迹观察RNF13对IRE1磷酸化的影响。结果:RNF13基因沉默效率在80%以上。RNF13基因沉默后明显抑制细胞凋亡;敲低RNF13的细胞可抵抗衣霉素以及毒胡萝卜素的诱导的细胞凋亡。Caspase-3是细胞凋亡的关键蛋白。敲低RNF13后caspase-3的活性形式明显降低(降低70%,P<0.001)。在加入衣霉素引起内质网应激的情况下,敲除RNF13的细胞XBP1的切割活性明显降低。敲除RNF13的细胞中IREl的磷酸化明显降低(降低90%,P<0.001)。结论:RNF13通过IRE1-XBP1信号通路调节细胞凋亡。
Objective： While studying ER stress,we found that Ring finger protein 13（RNF13） have some functions related to apoptosis. Here, we want to investigate the molecular mechanism that how RNF13 regulate apoptosis. We want to check the expression level of active caspase3, XBP1 splicing and IRE1 phosphorylation level after RNF13 was knockdown. Methods： RNA extraction were performed using si NC and si RNF13 cells. Cells were treated with Tunicamycin and MTT experiment was performed to measure cell growth. Caspase3 protein level were tested in RNF13 knockdown. Next, XBP1 cleavage and IRE1 phosphorylation were tested in control and RNF13 knockdown cells. Results： RNF13 stable knockdown cell line were obtained, the knockdown efficiency was good（80% reduction）. RNF13 knockdown cells were resistant to Tunicamycin and staurosporine induced apoptosis. The expression level cleavaged caspase 3 was reduced dramatically when RNF13 was knockdown compared with that of control cells, indicating reduced cell apoptosis in RNF13 depletion condition. RNF13 knockdown cells showed lower activity of XBP1 splicing. The phosporylation of IRE1 was dramatically reduced when RNF13 was knockdown. RNF13 depletion dramatically downregulated the IRE1-XBP1 signalling pathway. Conclusion： RNF13 regulates ER stress induced apoptosis through IRE1-XBP1 signal pathway.
出处
《现代生物医学进展》
CAS
2016年第5期801-805,共5页
Progress in Modern Biomedicine
基金
中国博士后科学基金资助项目(2013T60103
2012M520249)
