Li Yueying, Hu Yong, Che Lilong, Jia Junhai, Chen Min
Department of Physiology, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China.
Department of Neonatology, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200040, P.R. China.
Oncol Lett. 2016 Jun;11(6):3605-3610. doi: 10.3892/ol.2016.4450. Epub 2016 Apr 18.
Previous studies have demonstrated that the nuclear localization of ras homolog family member A (RhoA), with prominent concentration in the nucleolus, is a common feature in human cancer tissues and cancer cell lines. Although a previous study has demonstrated that the nuclear translocation of RhoA occurs via active transport, a process that occurs through importin α in a nuclear factor-κB-dependent manner, the mechanism, biological function and pathological meaning of the nucleolar residency of RhoA remain to be elucidated. As the cell nucleolus is the site of ribosome biosynthesis, the aim of the present study was to investigate the association between RNA synthesis and the nucleolar localization of RhoA, as well as the molecular mechanisms underlying the residency of RhoA in the nucleolus of HEp-2 (human larynx epithelial carcinoma) cells. Indirect immunofluorescence microscopy was used to evaluate the subcellular distribution of nuclear RhoA, and immunoblotting analysis was used to determine the total cellular protein level of RhoA. Consistent with the results of previous studies, untreated HEp-2 cells exhibited bright nucleolar staining, indicating an increased concentration of RhoA in the nucleoli. Treatment with actinomycin D for the inhibition of RNA synthesis caused a redistribution of RhoA from the nucleoli to the nucleoplasm with a speckled staining pattern. Immunoblotting revealed that neither the total cellular amount of RhoA nor the integrity of RhoA was affected by treatment with actinomycin D. In cells that were treated at a decreased concentration (0.05 mg/l) of actinomycin D, the redistribution of RhoA was reversible following the removal of the drug from the culture medium. However, this reversal was not observed at an increased drug concentration (1 mg/l). Overall, to the best of our knowledge, the results of the present study provide the first evidence that the inhibition of RNA synthesis induces a redistribution of nucleolar RhoA to the nucleoplasm, and additionally suggest that the nucleolar residency of RhoA in HEp-2 cells may be associated with active RNA synthesis.
先前的研究表明，ras同源家族成员A（RhoA）的核定位在核仁中高度富集，这是人类癌症组织和癌细胞系的一个共同特征。尽管先前的一项研究表明RhoA的核转位是通过主动运输发生的，这一过程通过输入蛋白α以核因子κB依赖的方式进行，但RhoA在核仁中的驻留机制、生物学功能和病理意义仍有待阐明。由于细胞核仁是核糖体生物合成的场所，本研究的目的是探讨RNA合成与RhoA核仁定位之间的关联，以及RhoA在人喉上皮癌细胞（HEp -2）核仁中驻留的分子机制。采用间接免疫荧光显微镜评估核RhoA的亚细胞分布，并采用免疫印迹分析确定细胞中RhoA的总蛋白水平。与先前的研究结果一致，未处理的HEp -2细胞呈现明亮的核仁染色，表明核仁中RhoA浓度增加。用放线菌素D处理以抑制RNA合成导致RhoA从核仁重新分布到核质，呈现斑点状染色模式。免疫印迹显示，放线菌素D处理既不影响细胞中RhoA的总量，也不影响RhoA的完整性。在以较低浓度（0.05 mg/l）放线菌素D处理的细胞中，从培养基中去除药物后，RhoA的重新分布是可逆的。然而，在较高药物浓度（1 mg/l）下未观察到这种逆转。总体而言，据我们所知本研究结果首次证明RNA合成抑制可诱导核仁RhoA重新分布到核质，此外还表明RhoA在HEp -2细胞中的核仁驻留可能与活跃的RNA合成有关。
