COPS3的稳定干涉细胞系建立及其在NLRP3炎症小体激活中的作用

COPS3(COP9 Signalosome Subunit 3)作为COP9信号复合体的第三个亚基,在多种恶性肿瘤中高表达。本实验室通过酵母双杂交系统筛选,发现COPS3与NLRP3 (NOD-like receptor family, pyrin domain containing 3)存在相互作用。目前关于COPS3与NLRP3炎症小体的相关性未见文献报道。为了研究COPS3对NLRP3炎症小体的影响,我们利用慢病毒感染系统建立了稳定干涉COPS3的THP1细胞系,首先将COPS3的不同干涉序列片段稳定整合到THP1细胞中,通过嘌呤霉素压力筛选,应用实时定量荧光PCR(Real time PCR)手段检测COPS3的干涉效果,最终确定成功建立干涉COPS3的稳定细胞株。在稳定敲低COPS3的细胞株中,我们发现NLRP3 炎症小体的激活受到明显抑制。综上提示,COPS3能够正向调节NLRP3炎症小体的激活。

Construction of THP1-COPS3-knock Down Stable Cell Line and Detection Effect of COPS3 on NLRP3 Inflammasome Activation

COPS3 is one subunit of the COP9 signalosome which is highly expressed in various malignant tumors. We found that COPS3 interacts with NLRP3 (NOD-like receptor family, pyrin domain containing 3) by yeast two-hybrid screening system. The correlation between COPS3 and NLRP3 inflammation is still unknown. To investigate the effects of COPS3 on NLRP3 inflammasome activation, we established a THP1 cell line with stable expression of COPS3 shRNA through lentivirus particles infection. We firstly integrated different COPS3 interfering sequences into THP1 cells through lentivirus vector PLKO.1 and then get the Anti-puro Positive cells under puromycin. The interference efficiency of COPS3 shRNA was detected via Real-Time PCR. Our results revealed that the activation of NLRP3 inflammasome is inhibited in COPS3 knock-down cell line compared with the control cells, suggesting that COPS3 may be a positive regulator of NLRP3 inflammasome.