Chinese Medical Sciences Journal ›› 2018, Vol. 33 ›› Issue (3): 143-151.doi: 10.24920/11815

• 论著 • 上一篇    下一篇

RNA结合蛋白UNR促进人神经胶质瘤细胞的迁移并调控核糖体蛋白L9的表达

田宁宇,齐颖娇,胡艳,阴彬,袁建刚,强伯勤,韩为(),彭小忠()   

  1. 中国医学科学院基础医学研究所 北京协和医学院基础学院,国家医学分子生物重点实验室,北京 100005
  • 出版日期:2018-09-30 发布日期:2018-06-01
  • 通讯作者: 韩为,彭小忠 E-mail:hanwei2012@ibms.pumc.edu.cn;pengxiaozhong@pumc.edu.cn

RNA-binding Protein UNR Promotes Glioma Cell Migration and Regulates the Expression of Ribosomal Protein L9

Tian Ningyu,Qi Yingjiao,Hu Yan,Yin Bin,Yuan Jiangang,Qiang Boqin,Peng Xiaozhong(),Han Wei()   

  1. State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences & School of Basic Medicine Peking Union Medical College, Beijing 100005, China
  • Published:2018-09-30 Online:2018-06-01
  • Contact: Peng Xiaozhong,Han Wei E-mail:hanwei2012@ibms.pumc.edu.cn;pengxiaozhong@pumc.edu.cn

摘要:

目的 研究RNA结合蛋白UNR在神经胶质瘤发生发展中的作用以及其发挥作用的分子机制。方法 首先,利用生物信息学方法分析CGGA数据库中UNR的表达水平及其表达水平高低与病人预后的关系。采用western blot和real-time PCR方法分析胶质瘤病人组织样本和细胞株中UNR的表达水平。其次,在胶质瘤细胞株A172,HS683,LN18中转染siRNA敲低UNR,采用MTS和划痕实验检测肿瘤细胞增殖能力以及迁移能力的变化。再次,通过分析已发布的黑色素瘤UNR iCLIP-seq、RNA-seq 和核糖体印迹测序数据库筛选出UNR在胶质瘤细胞中可能结合的靶mRNA。利用RNA免疫沉淀和生物素标记的RNA pull down实验确认UNR可结合核糖体蛋白L9(RPL9)的mRNA。最后,通过western blot检测RPL9在胶质瘤细胞株中的表达水平,并且检测敲低UNR以后RPL9蛋白水平的变化。在神经胶质瘤细胞株A172,T98G中转染siRNA敲低RPL9,检测和肿瘤侵袭迁移有关的上皮向间充质转化有关的标志物—波形蛋白的表达变化。结果 生物信息学分析UNR mRNA表达水平,发现其在恶性程度更高的胶质瘤样本中表达较高(P<0.001)。高表达UNR的患者预后较差(P=0.0177)。与正常细胞株以及正常脑组织样本相比,UNR在9株胶质瘤细胞以及患者组织样本中具有较高的表达水平(P<0.01)。敲低UNR抑制肿瘤细胞的迁移能力(P<0.05),但不影响其增殖能力。UNR可以结合在PTEN mRNA和RPL9 mRNA的3’非翻译区。敲低UNR以后RPL9的表达水平下调。RPL9在9株胶质瘤细胞中具有高表达的趋势,RPL9可以正向调控波形蛋白的表达。结论 本研究揭示了RNA结合蛋白UNR在胶质瘤细胞迁移方面的功能,并且发现UNR可以结合PTEN和RPL9 mRNA的3’非翻译区并调控RPL9的表达。这为我们更近一步研究胶质瘤的发生发展提供了新思路。

关键词: UNR, 神经胶质瘤, 迁移, 核糖体蛋白L9, 波形蛋白

Abstract:

Objective To investigate the role of RNA binding protein—upstream-of-N-Ras (UNR) in the development of glioma and its molecular mechanism.Methods First, bioinformatics analysis of CGGA database was performed to detect UNR expression level and prognosis of patients with glioma. Western blot and real-time PCR were used to detect UNR expression level in glioma cell lines and tissues. Next, UNR siRNAs were transfected in glioma cells, and MTS assay and scratch wound-healing assay were used to detect changes in cell proliferation and migration. Then, the candidate UNR target mRNAs were identified by analyzing the sequencing data of UNR iCLIP-seq, RNA sequencing and ribosome profiling databases of human melanoma. RNA immunoprecipitation and biotin pull-down assays were used to identify the UNR target mRNAs in glioma cells. Finally, western blot was used to detect the effect of UNR knockdown on ribosomal protein L9 (RPL9) and RPL9 protein expression level in glioma cell lines. RPL9 siRNA was transfected in A172 and T98G and the expression of vimentin in the cells was detected with western blot.Results Bioinformatics analysis showed that UNR mRNA expression level was significantly higher in high-grade glioma [Grade Ⅱ (n=126), Grade Ⅲ (n=51), Grade Ⅳ (n=128), P<0.001]. UNR high expression levels were associated with poor prognosis (P=0.0177). UNR had high expression level in glioma cell lines and patient samples compared with normal cell lines and normal brain samples (P<0.01). Knockdown of UNR inhibited glioma cells migration (P<0.05), but did not inhibit glioma cells growth in three glioma cell lines. UNR binded the 3’ untranslated region (UTR) of PTEN and RPL9 mRNAs. RPL9 protein was significantly highly expressed in most glioma cell lines (n=9) and knockdown of UNR resulted in a downregulation of RPL9 protein expression. Epithelial-mesenchymal transition (EMT)-related marker—vimentin was positively regulated by RPL9.Conclusions UNR could bind to the 3’UTR of PTEN and RPL9 in glioma cell lines, therefore promoting glioma cell migration and regulating the expression of RPL9. Here, we establish a link between UNR and RPL9 protein, which will provide new ideas for the further study of glioma.

Key words: UNR, glioma, migration, ribosomal protein L9, vimentin

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