抑制SHARPIN激活Rip1促进LNCaP-AI细胞坏死性凋亡  

Activation of Rip1 promotes necroptosis in LNCaP-AI cells via inhibiting SHARPIN

作　　者：王淦平[1] 黄海[1] 陈贤举 赖义明 林春浩[1] 曾乐祥[2] 曹亿[3] 张一鸣[4] 余永晟[5] 郭正辉[1] 

机构地区：[1]中山大学孙逸仙纪念医院泌尿外科,广东广州510120 [2]中山大学孙逸仙纪念医院小儿外科,广东广州510120 [3]中山大学孙逸仙纪念医院急诊外科,广东广州510120 [4]南方医科大学珠江医院泌尿外科,广东广州510282 [5]中山大学附属博济医院泌尿外科,广东广州511300

出　　处：《中国病理生理杂志》2016年第7期1214-1220,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81272807);广东省科技计划资助项目(No.2014A020212018);广东省医学研究资助项目(No.A2015113)

摘　　要：目的:探讨SHARPIN对去势抵抗性前列腺癌细胞LNCaP-AI坏死性凋亡关键因子Rip1的调控作用,以及对细胞坏死性凋亡的影响。方法:将LNCaP-AI细胞分为TNF-α+Z-VAD(caspase抑制剂)处理组与TNF-α+Z-VAD+Nec-1(Rip1抑制剂)处理组,应用MTS检测各组细胞的活力,研究坏死性凋亡机制在诱导LNCaP-AI细胞死亡中的作用。将LNCaP-AI细胞分为阴性对照组和SHARPIN干扰(si-SHARPIN)组,RT-qPCR验证抑制效率,通过免疫荧光等技术进一步探讨SHARPIN调控坏死性凋亡的具体分子机制。结果:与对照组相比,TNF-α+Z-VAD处理组的LNCaP-AI细胞活力下降28%(P<0.05),而TNF-α+Z-VAD+Nec-1处理组的细胞在Rip1被抑制后,细胞活力无明显改变。在LNCaP-AI细胞中,通过siRNA抑制SHARPIN表达后,Rip1表达水平上调,同时,LNCaP-AI细胞坏死性凋亡比例升高。结论:LNCaP-AI细胞可通过坏死性凋亡机制诱导死亡,下调SHARPIN可能通过激活Rip1增强LNCaP-AI细胞坏死性凋亡。AIM： To explore the role of SHARPIN in regulation of Rip1 in castration-resistant prostate cancer LNCaP-AI cells. METHODS： The LNCaP-AI cells were treated with TNF-α ＋ Z-VAD（ an inhibitor of pan-caspase） to activate necroptosis,which were compared to the cells treated with TNF-α ＋ Z-VAD ＋ Nec-1（ an inhibitor of Rip1）. A blank group and a TNF-α-treated group were set up as controls. The cell viability in each group was measured by MTS assay. In addition,SHARPIN was knocked down by siRNA,and the inhibitory efficiency was evaluated by RT-qPCR. The expression of Rip1 at mRNA and protein levels after knocking down SHARPIN was determined by RT-qPCR and Western blot to explore the underlying mechanism of regulatory network of necroptosis in prostate cancer. RESULTS： Compared with blank control group and TNF-α-treated group,the viability of LNCaP-AI cells treated with TNF-α ＋ Z-VAD decreased by 28%（ P〈 0. 05）. After treated with TNF-α ＋ Z-VAD ＋ Nec-1,the LNCaP-AI cells showed no significant difference in the viability compared with blank control and TNF-α-treated groups. Taken together,necroptosis may be an important way of cell death in LNCaP-AI cells. Besides,the expression of Rip1 at protein level was up-regulated following the inhibition of SHARPIN using siRNA,indicating that down-regulation of SHARPIN enhanced necroptosis via activating Rip1 in LNCaP-AI cells. CONCLUSION： Necroptosis is an important way of cell death. Inhibition of oncogenic factor SHARPIN enhances necroptosis via activating Rip1 in LNCaP-AI cells.

关 键 词：去势抵抗性前列腺癌 LNCaP-AI细胞 Rip1 SHARPIN 坏死性凋亡 

分 类 号：R363.2[医药卫生—病理学]