Journal: Cancers

Article Title: Downregulation of the Ubiquitin-E3 Ligase RNF123 Promotes Upregulation of the NF-κB1 Target SerpinE1 in Aggressive Glioblastoma Tumors

doi: 10.3390/cancers12051081

Figure Lengend Snippet: Analysis of cell proliferation and invasion in GB cells with RNF123-OV. ( A ) A172, HS683, and LN18 cell lines were stably transfected with empty vector (EV1) or a cDNA encoding Myc- RNF123 (RNF123-OV). RNF123-OV and p50 were assessed by Western blot, and β-actin was used as a loading control. ( B ) Quantification of RNF123 expression by RT-qPCR ( t -test, *** p < 0.001). ( C , D ) Proliferation of LN18 (C) and A172 (D) cell lines with RNF123-OV or the empty vector (EV1) (two-way ANOVA, Bonferroni correction *** p < 0.001). ( E , F ) Colony-forming units LN18 (E) and A172 (F) cell lines stably expressing control empty vector 1 (EV1) or RNF123-OV ( t -test, *** p < 0.001). ( G , H ) Percentage of invasion in LN18 (G) and A172 (H) cell lines stably expressing control (EV1) or RNF123-OV ( t -test, * p < 0.05, ** p < 0.01). ( I ) LN18 cell lines expressing control vector (EV1) or RNF123-OV were analyzed by RNA-sequencing to determine differentially expressed (DE) genes in RNF123-OV cell lines. The image shows a heatmap of the most DE genes (adjusted p < 0.05). ( J ) LN18 (RPPA1) and HS683 (RPPA2) cell lines with RNF123-OV were analyzed by RPPA. The image shows a heatmap of the most DE genes in RNF123-OV cell lines (adjusted p < 0.05). ( K ) Integration of DE genes identified in RPPA1 (LN18), RPPA2 (HS683), and RNA-sequencing in RNF123-OV cell lines that are targets of the NF-κB pathway. ( L ) RT-qPCR for SERPINE1 in A172, HS683, and LN18 cell lines expressing EV1 or RNF123-OV ( t -test, *** p < 0.001). ( M ) Western blot for SerpinE1 in A172, HS683, and LN18 cell lines expressing EV1 or RNF123-OV; low (L) and high (H) exposure times for the same image are shown. ( N ) Correlation analysis of RNF123 and SERPINE1 expression using the TCGA dataset from GB tumors ( n = 145; Spearman’s r = −0.27, p < 0.001). Error bars represent the mean ± SD from n = 3 replicates.

Article Snippet: To establish RNF123 overexpressing clones, LN18, A172, and HS683 cell lines (5 × 10 5 cells in 60 mm dishes) (Corning, NY, USA) were transfected with Myc-tagged RNF123 vector (OriGene, Rockville, MD) using the jetPRIME transfection reagent.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Control, Expressing, Quantitative RT-PCR, RNA Sequencing

Journal: Cancers

Article Title: Downregulation of the Ubiquitin-E3 Ligase RNF123 Promotes Upregulation of the NF-κB1 Target SerpinE1 in Aggressive Glioblastoma Tumors

doi: 10.3390/cancers12051081

Figure Lengend Snippet: MiR-155-5p decreased RNF123 expression and gave a poor prognosis in GB patients. ( A ) TCGA analysis of a merged cohort of low-grade glioma and GB for copy number variations and mutations. The frequency of RNF123 alteration is 0.8% of a total of 1084 patients. ( B ) Correlation analysis of miR-155-5p and RNF123 expression using TCGA dataset from GB tumors ( n = 145; Pearson r = −0.277, p = 0.0007). ( C ) TCGA database analysis of miR-155 in GB tissue ( n = 202) compared to normal brain tissue ( n = 11) ( t -test, *** p < 0.001). ( D ) Rembrandt database analysis of miR-155 expression in GB tissue ( n = 214) compared to normal brain tissue ( n = 21) ( t -test, *** p < 0.001). ( E ) TCGA database analysis of RNA-sequencing data for miR-155 in IDH WT ( n = 145) or mutated ( n = 8) GB tissue compared to normal brain tissue ( n = 5) (one-way ANOVA, *** p < 0.001, NS = non-significant). ( F ) LN18, A172, and HS683 cell lines were transfected with pre-miR-155-5p (miR-155-5p-OV) or miR control (miR-Ctrl) and RNF123 expression was quantified by Western blot. ( G ) miR-155-5p sequence aligned with human RNF123 WT 3′-UTR (WT) and RNF123 Mutant 3′-UTR (Mut) sequences. ( H ) Luciferase reporter activity assay to determine the effect of miR-155-5p on 3′-UTR of RNF123 using human RNF123 3′-UTR (WT) and RNF123 Mutant 3′-UTR (Mut) sequences cloned in RenSP vector ( t -test, NS = non-significant, *** p < 0.001). ( I ) Percentage of invasion of LN18 cell lines with miR-155-5p-OV, RNF123-OV, or both compared to control cell lines (one-way ANOVA, * p < 0.05, NS = non-significant). ( J ) Correlation analysis of miR-155-5p and SERPINE1 expression using TCGA dataset from GB tumors ( n = 145; Pearson r = 0.368, p < 0.0001). ( K ) GB patients from the TCGA database were split into low ( n = 90) and high ( n = 90) miR-155-5p expression and analyzed for RNF123 expression ( t -test, * p < 0.05). ( L ) Kaplan–Meier curves for the OS of GB patients expressing low ( n = 87) versus high ( n = 87) miR-155-5p (log-rank test, p = 0.024). ( M ) Dot plot to determine miR-155-5p expression in pre-operative plasma from GB patients ( n = 19) and plasma of healthy controls ( n = 46) ( t -test, ** p = 0.024). Error bars represent mean ± SD from replicates ( n = 3).

Article Snippet: To establish RNF123 overexpressing clones, LN18, A172, and HS683 cell lines (5 × 10 5 cells in 60 mm dishes) (Corning, NY, USA) were transfected with Myc-tagged RNF123 vector (OriGene, Rockville, MD) using the jetPRIME transfection reagent.

Techniques: Expressing, RNA Sequencing, Transfection, Control, Western Blot, Sequencing, Mutagenesis, Luciferase, Activity Assay, Clone Assay, Plasmid Preparation, Clinical Proteomics