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...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.展开更多
OBJECTIVE To explore the mechanism of translation initiation regulation of RPA2 via IRES by UNR and e IF3a.METHODS Biotin pull down assay was taken to study the binding of RPA2 IRES and UNR.UNR was knocked down and ov...OBJECTIVE To explore the mechanism of translation initiation regulation of RPA2 via IRES by UNR and e IF3a.METHODS Biotin pull down assay was taken to study the binding of RPA2 IRES and UNR.UNR was knocked down and overexpressed in H1299,A549 and SK-MES cell lines.Western blotting and real-time PCR were used to detect protein level and m RNA level respectively.CO-IP assay was conducted for the interaction of e IF3a and UNR.GST pul down assay was carried out to explore the exact domains.And the domains of e IF3a and UNR binding to RPA2 IRES were explored with EMSA assay.RESULTS NUR protein can bind to RPA2 IRES as well as e IF3a.UNR regulated the protein expression of RPA2 in H1299,A549 and SK-MES cells,and there was no change in RPA2 m RNA.UNR combined with e IF3a via the first domain of UNR and the first domain of e IF3a.UNR bound to RPA2 IRES with the first domain.And there was no sufficient evidence for the binding domain of e IF3a with RPA2 IRES yet.CONCLUSION UNR worked with eI F3a and co-regulate the RPA2 IRES activity and further regulate the expression of protein.This is the possible regulation mechanisms of cellular internal ribosomal entry site affect translation initiation.展开更多
基金Supported by the CAMS Innovation Fund for Medical Sciences(CIFMS,2016-I2M-1-001)
文摘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.
基金The project supported by National Natural Science Foundation of China(81573463)
文摘OBJECTIVE To explore the mechanism of translation initiation regulation of RPA2 via IRES by UNR and e IF3a.METHODS Biotin pull down assay was taken to study the binding of RPA2 IRES and UNR.UNR was knocked down and overexpressed in H1299,A549 and SK-MES cell lines.Western blotting and real-time PCR were used to detect protein level and m RNA level respectively.CO-IP assay was conducted for the interaction of e IF3a and UNR.GST pul down assay was carried out to explore the exact domains.And the domains of e IF3a and UNR binding to RPA2 IRES were explored with EMSA assay.RESULTS NUR protein can bind to RPA2 IRES as well as e IF3a.UNR regulated the protein expression of RPA2 in H1299,A549 and SK-MES cells,and there was no change in RPA2 m RNA.UNR combined with e IF3a via the first domain of UNR and the first domain of e IF3a.UNR bound to RPA2 IRES with the first domain.And there was no sufficient evidence for the binding domain of e IF3a with RPA2 IRES yet.CONCLUSION UNR worked with eI F3a and co-regulate the RPA2 IRES activity and further regulate the expression of protein.This is the possible regulation mechanisms of cellular internal ribosomal entry site affect translation initiation.
