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（英文）
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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 iCLIPseq、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m RNA。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。
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 siR NA was transfected in A172 and T98 G 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 highgrade 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.
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 siR NA was transfected in A172 and T98 G 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 highgrade 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.
引文
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6.Boussadia O,Niepmann M,Creancier L,et al.UNR is required in vivo for efficient initiation of translation from the internal ribosome entry sites of both rhinovirus and poliovirus.J Virol 2003;77(6):3353-9.
7.Cornelis S,Tinton SA,Schepens B,et al.UNR translation can be driven by an IRES element that is negatively regulated by polypyrimidine tract binding protein.Nucleic Acids Res 2005;33(10):3095-108.
8.Dormoy-Raclet V,Markovits J,Jacquemin-Sablon A,et al.Regulation of UNR expression by 5’-and 3’-untranslated regions of its mRNA through modulation of stability and IRES mediated translation.RNA Biol2005;2(3):e27-35.
9.Patel GP,Ma S,Bag J.The autoregulatory translational control element of poly(A)-binding protein mRNAforms a heteromeric ribonucleoprotein complex.Nucleic Acids Res 2005;33(22):7074-89.
10.Elatmani H,Dormoy-Raclet V,Dubus P,et al.The RNA-binding protein UNR prevents mouse embryonic stem cells differentiation toward the primitive endoderm lineage.Stem Cells 2011;29(10):1504-16.doi:10.1002/stem.712.
11.Dinur M,Kilav R,Sela-Brown A,et al.In vitro evidence that upstream of N-ras participates in the regulation of parathyroid hormone messenger ribonucleic acid stability.Mol Endocrinol 2006;20(7):1652-60.
12.Kobayashi H,Kawauchi D,Hashimoto Y,et al.The control of precerebellar neuron migration by RNA-binding protein Csde1.Neuroscience 2013;253:292-303.doi:10.1016/j.neuroscience.2013.08.055.
13.Ju Lee H,Bartsch D,Xiao C,et al.A post-transcriptional program coordinated by CSDE1 prevents intrinsic neural differentiation of human embryonic stem cells.Nat Commun 2017;8(1):1456.doi:10.1038/s41467-017-01744-5.
14.Horos R,Ijspeert H,Pospisilova D,et al.Ribosomal deficiencies in Diamond-Blackfan anemia impair translation of transcripts essential for differentiation of murine and human erythroblasts.Blood 2012;119(1):262-72.doi:10.1182/blood-2011-06-358200.
15.Xia K,Guo H,Hu Z,et al.Common genetic variants on 1p13.2 associate with risk of autism.Mol Psychiatry 2014;19(11):1212-9.doi:10.1038/mp.2013.146.
16.Wurth L,Papasaikas P,Olmeda D,et al.UNR/CSDE1drives a post-transcriptional program to promote melanoma invasion and metastasis.Cancer Cell 2016;30(5):694-707.doi:10.1016/j.ccell.2016.10.004.
17.Martinez-Useros J,Georgiev-Hristov T,Fernandez-Acenero MJ,et al.UNR/CDSE1 expression as prognosis biomarker in resectable pancreatic ductal adenocarcinoma patients:a proof-of-concept.PLoSOne 2017;12(8):e0182044.doi:10.1371/journal.pone.0182044.
18.Hu PS,Xia QS,Wu F,et al.NSPc1 promotes cancer stem cell self-renewal by repressing the synthesis of all-trans retinoic acid via targeting RDH16 in malignant glioma.Oncogene 2017;36(33):4706-18.doi:10.1038/onc.2017.34.
19.Ray S,Anderson EC.Stimulation of translation by human Unr requires cold shock domains 2 and 4,and correlates with poly(A)binding protein interaction.Sci Rep 2016;6:22461.doi:10.1038/srep22461.
20.Dormoy-Raclet V,Markovits J,Malato Y,et al.UNR,a cytoplasmic RNA-binding protein with cold-shock domains,is involved in control of apoptosis in ES and HuH7 cells.Oncogene 2007;26(18):2595-605.
21.Tang Z,Li C,Kang B,et al.GEPIA:a web server for cancer and normal gene expression profiling and interactive analyses.Nucleic Acids Res 2017;45(W1):W98-W102.doi:10.1093/nar/gkx247.
22.Zhang Y,Chao T,Li R,et al.MicroRNA-128 inhibits glioma cells proliferation by targeting transcription factor E2F3a.J Mol Med(Berl)2009;87(1):43-51.doi:10.1007/s00109-008-0403-6.
23.Legler JM,Ries LA,Smith MA,et al.Cancer surveillance series[corrected]:brain and other central nervous system cancers:recent trends in incidence and mortality.J Natl Cancer Inst 1999;91(16):1382-90.
2.Campos-Melo D,Droppelmann CA,Volkening K,et al.RNA-binding proteins as molecular links between cancer and neurodegeneration.Biogerontology 2014;15(6):587-610.doi:10.1007/s10522-014-9531-2.
3.Han W,Xin Z,Zhao Z,et al.RNA-binding protein PCBP2 modulates glioma growth by regulating FHL3.J Clin Invest 2013;123(5):2103-18.doi:10.1172/JCI61820.
4.Jeffers M,Paciucci R,Pellicer A.Characterization of UNR:a gene closely linked to N-ras.Nucleic Acids Res1990;18(16):4891-9.
5.Hunt SL,Hsuan JJ,Totty N,et al.UNR,a cellular cytoplasmic RNA-binding protein with five cold-shock domains,is required for internal initiation of translation of human rhinovirus RNA.Genes Dev 1999;13(4):437-48.
6.Boussadia O,Niepmann M,Creancier L,et al.UNR is required in vivo for efficient initiation of translation from the internal ribosome entry sites of both rhinovirus and poliovirus.J Virol 2003;77(6):3353-9.
7.Cornelis S,Tinton SA,Schepens B,et al.UNR translation can be driven by an IRES element that is negatively regulated by polypyrimidine tract binding protein.Nucleic Acids Res 2005;33(10):3095-108.
8.Dormoy-Raclet V,Markovits J,Jacquemin-Sablon A,et al.Regulation of UNR expression by 5’-and 3’-untranslated regions of its mRNA through modulation of stability and IRES mediated translation.RNA Biol2005;2(3):e27-35.
9.Patel GP,Ma S,Bag J.The autoregulatory translational control element of poly(A)-binding protein mRNAforms a heteromeric ribonucleoprotein complex.Nucleic Acids Res 2005;33(22):7074-89.
10.Elatmani H,Dormoy-Raclet V,Dubus P,et al.The RNA-binding protein UNR prevents mouse embryonic stem cells differentiation toward the primitive endoderm lineage.Stem Cells 2011;29(10):1504-16.doi:10.1002/stem.712.
11.Dinur M,Kilav R,Sela-Brown A,et al.In vitro evidence that upstream of N-ras participates in the regulation of parathyroid hormone messenger ribonucleic acid stability.Mol Endocrinol 2006;20(7):1652-60.
12.Kobayashi H,Kawauchi D,Hashimoto Y,et al.The control of precerebellar neuron migration by RNA-binding protein Csde1.Neuroscience 2013;253:292-303.doi:10.1016/j.neuroscience.2013.08.055.
13.Ju Lee H,Bartsch D,Xiao C,et al.A post-transcriptional program coordinated by CSDE1 prevents intrinsic neural differentiation of human embryonic stem cells.Nat Commun 2017;8(1):1456.doi:10.1038/s41467-017-01744-5.
14.Horos R,Ijspeert H,Pospisilova D,et al.Ribosomal deficiencies in Diamond-Blackfan anemia impair translation of transcripts essential for differentiation of murine and human erythroblasts.Blood 2012;119(1):262-72.doi:10.1182/blood-2011-06-358200.
15.Xia K,Guo H,Hu Z,et al.Common genetic variants on 1p13.2 associate with risk of autism.Mol Psychiatry 2014;19(11):1212-9.doi:10.1038/mp.2013.146.
16.Wurth L,Papasaikas P,Olmeda D,et al.UNR/CSDE1drives a post-transcriptional program to promote melanoma invasion and metastasis.Cancer Cell 2016;30(5):694-707.doi:10.1016/j.ccell.2016.10.004.
17.Martinez-Useros J,Georgiev-Hristov T,Fernandez-Acenero MJ,et al.UNR/CDSE1 expression as prognosis biomarker in resectable pancreatic ductal adenocarcinoma patients:a proof-of-concept.PLoSOne 2017;12(8):e0182044.doi:10.1371/journal.pone.0182044.
18.Hu PS,Xia QS,Wu F,et al.NSPc1 promotes cancer stem cell self-renewal by repressing the synthesis of all-trans retinoic acid via targeting RDH16 in malignant glioma.Oncogene 2017;36(33):4706-18.doi:10.1038/onc.2017.34.
19.Ray S,Anderson EC.Stimulation of translation by human Unr requires cold shock domains 2 and 4,and correlates with poly(A)binding protein interaction.Sci Rep 2016;6:22461.doi:10.1038/srep22461.
20.Dormoy-Raclet V,Markovits J,Malato Y,et al.UNR,a cytoplasmic RNA-binding protein with cold-shock domains,is involved in control of apoptosis in ES and HuH7 cells.Oncogene 2007;26(18):2595-605.
21.Tang Z,Li C,Kang B,et al.GEPIA:a web server for cancer and normal gene expression profiling and interactive analyses.Nucleic Acids Res 2017;45(W1):W98-W102.doi:10.1093/nar/gkx247.
22.Zhang Y,Chao T,Li R,et al.MicroRNA-128 inhibits glioma cells proliferation by targeting transcription factor E2F3a.J Mol Med(Berl)2009;87(1):43-51.doi:10.1007/s00109-008-0403-6.
23.Legler JM,Ries LA,Smith MA,et al.Cancer surveillance series[corrected]:brain and other central nervous system cancers:recent trends in incidence and mortality.J Natl Cancer Inst 1999;91(16):1382-90.