ARRB1-Mediated Regulation of E2F Target Genes in Nicotine-Induced Growth of Lung Tumors
ARRB1-Mediated Regulation of E2F Target Genes in Nicotine-Induced Growth of Lung Tumors
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DOI：
10.1093/jnci/djq541
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发表时间：
2011-02-01
影响因子：
10.3
通讯作者：
Chellappan, Srikumar P.
中科院分区：
文献类型：
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作者：
Dasgupta, Piyali;Rizwani, Wasia;Chellappan, Srikumar P.
背景尼古丁通过尼古丁乙酰胆碱受体和抑制蛋白β 1（ARRB 1）蛋白诱导非小细胞肺癌（NSCLC）细胞增殖。然而，是否ARRB 1易位到细胞核后，烟碱乙酰胆碱受体激活和它如何调节人类NSCLC的生长是未知的。方法我们研究了ARRB 1在人类NSCLC细胞系（A549和H1650），正常肺细胞系（NHBE和SAEC），和肺癌组织芯片的核定位。用ARRB 1特异性短发夹RNA（A549-sh）转染A549细胞以敲低ARRB 1表达，或用空载体（A549-EV）转染A549细胞以检查ARRB 1在尼古丁的促有丝分裂和抗凋亡作用中的作用，ARRB 1与E2 F转录因子的结合，以及ARRB 1在尼古丁诱导的E2 F调节的存活和增殖基因细胞分裂周期6同源物（CDC 6），胸苷酸合成酶（TYMS），和含有杆状病毒IAP重复序列的5（BIRC 5）。采用实时荧光定量聚合酶链反应技术对mRNA表达进行定量分析。对A549细胞和新鲜冷冻的人NSCLC肿瘤（n = 8）进行染色质免疫沉淀测定，以检查ARRB 1、E1 A结合蛋白（EP 300）和乙酰化组蛋白3（Ac-H3）对E2 F调节基因的结合。结果尼古丁可诱导非小细胞肺癌和正常肺组织中ARRB 1的核转位，吸烟者肺癌组织中ARRB 1的核定位增强。在A549-sh细胞中，尼古丁的促有丝分裂和抗凋亡作用降低。在正常肺细胞、NSCLC细胞和肿瘤中，核ARRB 1与E2 F转录因子结合。尼古丁处理诱导A549-EV细胞中E2 F调控基因的表达显著增加，但在A549-sh细胞中没有;在BIRC 5中观察到的最大差异（A549-EV vs A549-sh，尼古丁处理后mRNA水平的平均增加倍数= 20.7倍，95%置信区间= 19.2- 22.2倍，vs平均值= 0.8倍，95%置信区间= 0.78至0.82倍，P < .001）。结论尼古丁可诱导ARRB 1核转位，促进增殖和存活相关基因的表达，从而促进NSCLC的生长和发展。
Background Nicotine induces the proliferation of non-small cell lung cancer (NSCLC) cells via nicotinic acetylcholine receptors and the arrestin, beta 1 (ARRB1) protein. However, whether ARRB1 translocates to the nucleus upon nicotinic acetylcholine receptor activation and how it regulates growth of human NSCLCs are not known.Methods We investigated nuclear localization of ARRB1 in human NSCLC cell lines (A549 and H1650), normal lung cell lines (NHBE and SAEC), and lung cancer tissue microarray. A549 cells were transfected with ARRB1-specific short hairpin RNA (A549-sh) to knockdown ARRB1 expression, or with empty vector (A549-EV), to examine the role of ARRB1 in the mitogenic and antiapoptotic effects of nicotine, binding of ARRB1 to E2F transcription factors, and the role of ARRB1 in nicotine-induced expression of E2F-regulated survival and proliferative genes cell division cycle 6 homolog (CDC6), thymidylate synthetase (TYMS), and baculoviral IAP repeat-containing 5 (BIRC5). Real-time polymerase chain reaction was performed for quantitative analysis of mRNA expression. Chromatin immunoprecipitation assays were performed on A549 cells and fresh-frozen human NSCLC tumors (n = 8) to examine the binding of ARRB1, E1A binding protein (EP300), and acetylated histone 3 (Ac-H3) on the E2F-regulated genes. All statistical tests were two-sided.Results Nicotine induced the nuclear translocation of ARRB1 in NSCLC and normal lung cells, and lung tumor tissues from smokers showed an increased nuclear localization. The mitogenic and antiapoptotic effects of nicotine were reduced in A549-sh cells. Nuclear ARRB1 bound to E2F transcription factors in normal lung cells, NSCLC cells, and tumors. Nicotine treatment induced a statistically significant increased expression of E2F-regulated genes in A549-EV but not in A549-sh cells; the maximum difference being observed in BIRC5 (A549-EV vs A549-sh, mean fold-increase in mRNA level upon nicotine treatment = 20.7-fold, 95% confidence interval = 19.2- to 22.2-fold, vs mean = 0.8-fold, 95% confidence interval= 0.78- to 0.82-fold, P < .001). Furthermore, nicotine induced the binding of ARRB1, EP300, and Ac-H3 on E2F-regulated genes.Conclusion Nicotine induced the nuclear translocation of ARRB1 and showed increased expression of proliferative and survival genes, thereby contributing to the growth and progression of NSCLCs.