Fig 1: Selection of lncRNAs related to FUBP1 in chordoma (A) Cases in GSE98021 were divided into high FUBP1 group and low FUBP1 group using the median value of FUBP1 expression as cut-off; a total of 24 lncRNAs (ABCC13, ALDOAP2, CROCCP3, FAM138B, GAPDHP62, GEMIN8P4, GUSBP5, JMJD1C-AS1, KRT8P41, LOXL1-AS1, MNX1-AS1, NME2P1, OR10D1P, OR1F2P, PFN1P2, PGAM1P4, PLAC4, PYY2, RPS10P7, RPS2P32, SCAND2P, TCF4-AS1, TPI1P2, TPM3P9) were found to be significantly positively correlated with FUBP1. (B) The expression of these 24 lncRNAs was determined in a total of 5 paired chordoma and non-cancerous tissue samples using real-time qPCR. (C–E) The expression of lncRNA KRT8P41, TBXT and KRT8 was determined in 16 non-cancerous and 36 chordoma tissue samples using real-time qPCR. (F) The correlations of lncRNA KRT8P41between TBXT or KRT8 in tissue samples were analyzed using Spearman's rank correlation coefficient analysis. (G) The area under the receiver operating characteristic (ROC) curve (AUC) was calculated using the expression levels of lncRNA KRT8P41. (H) The survival percentage of chordoma patients with high or low lncRNA KRT8P41 expression was analyzed using a log-rank test and shown as Kaplan-Meier curves. **P < 0.01.
Fig 2: LncRNA KRT8P41/miR-193a axis modulates chordoma cell aggressiveness through FUBP1 U-CH1 and JHC7 cells were co-transfected with miR-193a inhibitor and si-KRT8P41 and examined for (A and B) cell viability by MTT assay; (C) cell invasion by Transwell assay; (D and E) the protein levels of FUBP1 and c-Myc by Immunoblotting. **P < 0.01, compared with the control group; ##P < 0.01, compared with the si-NC + miR-193a inhibitor group.
Fig 3: miR-193a binds to lncRNA KRT8P41 and the 3'UTR of FUBP1 (A and C) The schematic diagrams showing the predicted miR-193a binding site in lncRNA KRT8P41 or the 3'UTR of FUBP1 and the structures of wt/mut-KRT8P41 and wt/mut-FUBP1 3'UTR luciferase reporter plasmids. (B and D) Luciferase reporter plasmids were co-transfected in 293 T cells with miR-193a mimics/inhibitor. The luciferase activity was determined. (E-F) RIP assays were performed to confirm miR-193a binding to lncRNA KRT8P41 and FUBP1 3'-UTR using IgG or AGO2 antibody. The levels of lncRNA KRT8P41, miR-193a, and FUBP1 in precipitated IgG or AGO2 proteins were examined using real-time PCR. **P < 0.01.
Fig 4: FUBP1 binds broadly to the Myc gene in parallel to the expression of MYC and loss of FUBPs causes changes in the chromatin at the Myc locus.a First panel: the FUBP1 ChIP-seq at Myc locus in MEFs. Peaks called by the SICER algorithm against input control are marked with green lines, FDR < 0.01. A mouse FUSE (mFUSE) with a high possibility of melting is marked by a pink line. The positions of the primers used in each panel of b and c were marked with the corresponding letters in this figure. The DNase hypersensitivity sites (DHS) downstream of Myc that were flanked by H3K4me1 peaks and overlapped with p300 peaks84 are marked by a light green rectangle and analyzed in d. Second to the fifth lines: the DHS peaks identified by TACh-seq in WT and KOKD MEFs, biological duplicates. Sixth panel: ENCODE data (ENCFF592HMA) of H3K4me1 ChIP-seq in MEFs. Seventh panel: p300 ChIP-seq in MEFs from published literature. Bottom panel: the transition probabilities calculated by SIDD algorithm as functions of base pair at the Myc locus and regions susceptible to melting are shown. The sequences of 3.7 kb upstream of the transcription start site (TSS) to the 1.2 kb downstream of TTS was analyzed as a superhelical domain, whereas 1.2 to 10 kb downstream of TTS was analyzed as another superhelical domain. Superhelical density = -0.06. The positions of the primers used in each panel of b and c were marked with the corresponding letters. b FUBP1 ChIP-qPCR results of the Myc locus in steady-state and PDGF- or TNFa-stimulated MEFs are shown. The positions of the primers used in each panel are marked with the corresponding letters in a. Experiments were performed in triplicate. **p < 0.01, T-test assuming unequal variance, two-tailed, compared to the serum-starved sample. c The results of H3K4me1 ChIP-qPCR on the Myc gene in WT, KD, KO, and KOKD MEFs are shown. The position of the primers used in this figure is marked with the corresponding letters in Figure a. Experiments were performed in triplicate. *p < 0.05, **p < 0.01, T-test assuming unequal variance, two-tailed, compared to the WT. d The average of the normalized TACh-seq reads density of two biological replicates of WT and KOKD MEFs were plotted on the DHS peak downstream of Myc (the region marked by a light green rectangle in a). DESeq2 analysis showed the signal of this DHS peak in KOKD is significantly higher in KOKD than that in WT (FDR < 0.05), with an average fold increase of 1.6.
Fig 5: Expression and correlation of lncRNA KRT8P41, miR-193a, and FUBP1 in tissue samples (A and B) The expression levels of miR-193a and FUBP1 were examined in 16 non-cancerous and 36 chordoma tissue samples using real-time qPCR. **P < 0.01. (C) The correlations of lncRNA KRT8P41, miR-193a, and FUBP1 in tissue samples were analyzed using Spearman's rank correlation coefficient analysis.
Supplier Page from Abcam for Anti-FUBP1/FBP antibody [EPR12327]