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Figure 1.

Upstream-of-N-Ras (UNR) is upregulated in glioma cell lines and tissues compared with normal human astrocytes and brain tissues. A. UNR mRNA expression level in whole genome gene profiling of glioma samples from the Chinese Glioma Genome Atlas (CGGA, http://www.cgcg.org.cn); Grade Ⅱ (n=126), Grade Ⅲ (n=51), Grade Ⅳ (n=128) (*P<0.05, **P<0.005, ***P<0.001, Student’s t-test). B. Kaplan-Meier curves showing high expression levels of UNR correlated with poor patient survival according to the CGGA database. C. Real-time PCR analysis of the relative UNR mRNA expression level in normal human brain (n=5), Grade Ⅱ (n=11), Grade Ⅲ (n=20), and Grade Ⅳ (n=32) glioma tissues (*P<0.05, ***P<0.001, Student’s t-test). D. Relative UNR protein levels in normal brain (n=7), Grade Ⅱ (n=12), Grade Ⅲ (n=24), and Grade Ⅳ (n=47) glioma tissues analyzed by western blot (*P<0.05, **P<0.01, Student’s t-test). E. Western blot showing the relative UNR protein expression level in 3 normal human astrocyte cell lines and 9 glioma cell lines."

Figure 2.

Knockdown of UNR inhibits glioma cell migration. A. Western blot analysis of UNR expression in 3 glioma cell lines (A172, LN18 and HS683) transiently transfected with the control siRNA or UNR siRNA for 48 hours. β-Actin was used as a loading control. B. MTS assay on 3 glioma cell lines after transfection with the control siRNA or UNR siRNA as above. The data have no statistical significance compared with the control siRNA. C, D. Wound-healing assays in HS683 and LN18 cells were performed after infection with control siRNA and UNR-siRNA for 0-72 hours. The assays in A172 cells were performed after infection with control siRNA and UNR-siRNA for 0-36 hours. Pictures were taken during this period. The graph represents the width of the remaining open wound calculated in relation to time 0 (*P<0.05 compared with control siRNA, student’s t-test). All samples were treated in triplicate in each experiment. Each assay was repeated three times."

Figure 3.

UNR binds 3’UTR of biotin-labeled candidate mRNAs. A. Real-time PCR of the candidate mRNAs by RNA binding protein immunoprecipitation of UNR antibodies in LN18 cell lysates. B. Schematic of the indicated candidate mRNAs. The red fragments represent the iCLIP-seq sequence, and the black fragments represent the probe sequences that were designed between 100 bp and 500 bp for the biotin pull-down assay to protect their secondary binding structures. C. Biotin pull-down analysis of complexes formed in vitro using biotin-labelled candidate mRNA segments (shown in B) in LN18 cell lysates."

Figure 4.

Downregulation of UNR inhibits the expression of RPL9 and RPL9 has high expression levels in gliomas. A. Knockdown of UNR inhibits the expression of RPL9 in the glioma cell lines assayed by western blot. B. RPL9 has a higher expression level in gliomas than in normal tissues in the TCGA and GTEx projects by using the GEPIA website (GBM: glioblastoma multiform, LGG: low grade glioma). Red: glioma patients. Gray: normal patients. C. Western blot showing the RPL9 protein levels in normal human astrocyte and glioma cell lines. D. Western blot analysis of RPL9 and vimentin expression in A172 and T98G cells transiently transfected with the control siRNA (SiNC) or RPL9 siRNA for 48 hours."