SHANK蛋白RH区域交互作用蛋白通过核转录因子通路上调生存素抑制前列腺癌细胞凋亡  

A preliminary study of SHANK-associated RH domain interacting protein decreasing apoptosis by nuclear factor kappa B pathway increased Survivin in prostate cancer cell lines

作　　者：黄海[1] 张一鸣[1] 杜涛[1] 何旺[1] 董文[1] 朱定军[1] 赖义明 刘皓[1] 于浩[1] 毕良宽[1] 林天歆[1] 黄健[1] 郭正辉[1] 

机构地区：[1]中山大学孙逸仙纪念医院泌尿外科,广州510120

出　　处：《中华实验外科杂志》2015年第4期827-830,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81101947、81272807、81472382）;中央高校基本科研业务费专项资金资助项目（14ykpy19）;广东省科技社会发展项目（201313021800107）

摘　　要：目的 探讨SHANK蛋白RH区域交互作用蛋白（SHARPIN）对前列腺癌细胞凋亡调控机制.方法 设计并合成抑制SHARPIN的小干扰RNA 3对,实时荧光定量聚合酶链反应法（FQ-PCR）筛选抑制效率最高者;Western blot验证对前列腺癌细胞中SHARPIN表达的抑制效果.Western blot检测各组中SHARPIN、生存素（Survivin）表达和核转录因子-κB（NF-κB）磷酸化情况;四唑氮化合物（MTS）法检测增殖能力;流式细胞仪检测细胞凋亡.结果 FQ-PCR显示第3对序列对SHARPIN抑制效率最高;Western blot检测显示其对3个前列腺癌细胞株中SHARPIN表达抑制效率均达70％以上.当SHARPIN被抑制后,p65的激活明显受抑,15 min时对p-p65抑制率达95.0％;Survivin的表达比对照组明显下调,加入NF-κB抑制剂后,Survivn表达量虽然也明显下降,但是远没有抑制SHARPIN后表达量下降的明显.抑制SHARPIN后,LNCap、DU145和PC-3的增殖抑制率分别为31.0％、56.8％和54.6％.抑制SHARPIN后,3种前列腺癌细胞早期凋亡明显增加.结论 SHARPIN可以通过NF-κB通路抑制前列腺癌细胞凋亡;Survivin可能是其并不唯一的下游调控因子.Objective To investigate SHANK-associated RH domain interacting protein （SHARPIN） regulation mechanism of apoptosis on prostate cancer cell lines.Methods First,we design and synthesize 3 pairs of small interfering RNA （siRNA） to inhibit SHARPIN,and real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） was used to screen the highest suppression efficiency siRNA.Western blotting was used to verify the inhibitory effect of SHARPIN on protein level in prostate cancer cell lines.Western blotting was used to detect the expressions of SHARPIN of each group,Survivin expression and phosphorylation of nuclear factor-κB （NF-κB）,to verify activation of SHARPIN on NF-κB pathway,and SHARPIN whether through NF-κB for Survivin regulation.3-（4,5-Dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium （MTS） method was used to detect the proliferation of prostate cancer cell lines.Flow cytometrywas used to verify the apoptosis in the groups.Results RT-PCR showed the third pair of siRNA was the most efficient sequence of SHARPIN inhibition,and western blot analysis showed that SHARPIN expression inhibition efficiency reached more than 70% in the three prostate cancer cell lines.When SHARPIN was inhibited,p65 activation was inhibited,and the highest inhibition rate was 95.0% at 15 min.Survivin expression was significantly lower than the control group,and after the NF-κB inhibitor was added,Survivin although the expression level was significantly declined,but far from the inhibition of expression of SHARPIN.After the suppression of SHARPIN,proliferation inhibition rates of LNCap,DU145 and PC-3 were 31.0%,56.8% and 54.6%,respectively.After the suppression of SHARPIN,the early apoptotic proportion was significantly increased in three PCa cell lines.Conclusion SHARPIN can restrain the apoptosis via NF-κB pathway in prostate cancer cell lines and Survivin is not only but possible downstream regulatory factors.

关 键 词：前列腺癌 SHANK蛋白RH区域交互作用蛋白 核转录因子 生存素 凋亡 

分 类 号：R737.25[医药卫生—肿瘤]