CAJ | 학술논문

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.
목적 탐토SHANK단백RH구역교호작용단백(SHARPIN)대전렬선암세포조망조공궤제.방법 설계병합성억제SHARPIN적소간우RNA 3대,실시형광정량취합매련반응법(FQ-PCR)사선억제효솔최고자;Western blot험증대전렬선암세포중SHARPIN표체적억제효과.Western blot검측각조중SHARPIN、생존소(Survivin)표체화핵전록인자-κB(NF-κB)린산화정황;사서담화합물(MTS)법검측증식능력;류식세포의검측세포조망.결과 FQ-PCR현시제3대서렬대SHARPIN억제효솔최고;Western blot검측현시기대3개전렬선암세포주중SHARPIN표체억제효솔균체70％이상.당SHARPIN피억제후,p65적격활명현수억,15 min시대p-p65억제솔체95.0％;Survivin적표체비대조조명현하조,가입NF-κB억제제후,Survivn표체량수연야명현하강,단시원몰유억제SHARPIN후표체량하강적명현.억제SHARPIN후,LNCap、DU145화PC-3적증식억제솔분별위31.0％、56.8％화54.6％.억제SHARPIN후,3충전렬선암세포조기조망명현증가.결론 SHARPIN가이통과NF-κB통로억제전렬선암세포조망;Survivin가능시기병불유일적하유조공인자.
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.