β-抑制蛋白1通过促进IL-6基因启动子区乙酰化调控非小细胞肺癌的细胞增殖和凋亡  

β-arrestin 1 facilitates acetylation of IL-6 gene promoter to affect proliferation and apoptosis of non-small cell lung cancer cells

作　　者：郑玄 王晓强[1] 刘文会 赵鑫鑫 杨斌 ZHENG Xuan;WANG Xiaoqiang;LIU Wenhui;ZHAO Xinxin;YANG Bin(Department of Thoracic Surgery,Tangshan Union Medical College Hospital,Tangshan 063000,Hebei Province,China;Department of Pediatric,Tangshan Union Medical College Hospital,Tangshan 063000,Hebei Province,China;Department of Surgery,Tangshan Union Medical College Hospital,Tangshan 063000,Hebei Province,China)

机构地区：[1]唐山市协和医院胸外科,河北唐山063000 [2]唐山市协和医院儿科,河北唐山063000 [3]唐山市协和医院外科,河北唐山063000

出　　处：《肿瘤》2020年第5期329-338,共10页Tumor

摘　　要：目的 :探讨β-抑制蛋白1(β-arrestin1,ARRB1)对非小细胞肺癌细胞增殖和凋亡的影响,并研究其可能的分子作用机制。方法 :在非小细胞肺癌细胞系A549和H1650中,通过转染小干扰RNA的方法敲低ARRB1基因表达,或通过转入Flag标签的ARRB1基因重组质粒使ARRB 1基因过表达,实时荧光定量PCR和蛋白质印迹法验证转染效果。采用实时荧光定量PCR和ELISA法分别检测干扰和过表达ARRB1对白细胞介素6(interleukin-6,IL-6)m RNA转录和分泌水平的影响。采用蛋白免疫共沉淀法检测ARRB1与IL-6关键调控分子p300的相互作用。采用染色质免疫共沉淀法检测干扰或过表达ARRB1对IL-6基因启动子区p300富集和组蛋白乙酰化修饰的影响。采用CCK-8法和FCM法分别检测干扰ARRB1表达对非小细胞肺癌A549细胞增殖和凋亡的影响。采用蛋白质印迹检测干扰或过表达ARRB1对IL-6/信号转导与转录激活因子3(signal transducer and activator of transcription 3,STAT3)信号通路相关分子活化和表达的影响。结果 :在非小细胞肺癌细胞系A549和H1650中有效敲低或过表达ARRB1后发现,ARRB1可促进IL-6的转录和分泌(P值均<0.05);正向和反向蛋白免疫共沉淀均检测到ARRB1与p300相互作用;同时发现ARRB1能促进p300在IL-6基因启动子区的富集(P值均<0.01),提高IL-6基因启动子区的乙酰化水平(P值均<0.05),促进非小细胞肺癌细胞增殖(P值均<0.01),抑制细胞凋亡。另外,ARRB1能提高STAT3磷酸化活化水平以及c-Myc和Bcl-2的表达水平。结论 :在非小细胞肺癌细胞中,ARRB1能够与p300相互作用,并促进p300向IL-6基因启动子区募集,从而提高IL-6基因启动子区组蛋白H3和H4的乙酰化水平,诱导IL-6的转录活化和分泌,并进一步触发IL-6/STAT3信号通路转导,提高STAT3磷酸化活化水平,使下游c-Myc和Bcl-2表达量增加,从而促进细胞增殖并抑制细胞凋亡。Objective: To investigate the effects of β-arrestin 1(ARRB1) on apoptosis and proliferation of non-small cell lung cancer(NSCLC) cells, and to explore the underlying molecular mechanism.Methods: In NSCLC cell lines A549 and H1650, the expression of ARRB1 was knocked down by transfection of siRNA or over-expressed by transfection of Flag-ARRB1 recombinant plasmids, which was verified by real-time fluorescent quantitative PCR and Western blotting. The effects of down-regulating and up-regulating ARRB1 expression on the transcription and secretion of interleukin-6(IL-6) were detected by real-time fluorescent quantitative PCR and ELISA method, respectively. The interaction between ARRB1 and p300 was detected by protein immunocoprecipitation. The recruitment of p300 and the acetylation of histone in IL-6 promoter region after ARRB1 knock-down or overexpression were detected by chromatin immunocoprecipitation. The proliferation and apoptosis of ARRB1 silencing A549 cells were detected by CCK-8 assay and FCM, respectively. The expression and activation of IL-6/signal transducer and activator of transcription 3(STAT3) signaling pathway-related molecules in ARRB1 silencing or overexpression NSCLC cells were investigated by Western blotting.Results: In ARRB1-silenced or overexpressed NSCLC cell lines A549 and H1650, ARRB1 enhanced the transcription and production of IL-6(all P < 0.05). The interaction of ARRB1 and p300 was confirmed by forward and reverse immunocoprecipitation. After ARRB1 knockdown or overexpression, it was found that ARRB1 enhanced the recruitment of p300 in IL-6 promoter region(both P < 0.01) and increased the acetylating of IL-6 promoter(both P < 0.05). Moreover, ARRB1 could facilitate the growth(P < 0.01) and apoptosis inhibition of NSCLC cells. ARRB1 could promote the phosphorylation of STAT3 and the expressions of c-Myc and Bcl-2 proteins.Conclusion: In NSCLC cells, ARRB1 interacts with p300, facilitates the recruitment of p300 to IL-6 promoter, and up-regulates the acetylation of histone H3 and H4

关 键 词：癌 非小细胞肺 β-抑制蛋白1 白细胞介素6 乙酰化作用 信号转导及转录激活因子3 

分 类 号：R730.26[医药卫生—肿瘤]