Journal: Cancer Research

Article Title: Aberrant Nuclear Export of circNCOR1 Underlies SMAD7-Mediated Lymph Node Metastasis of Bladder Cancer

doi: 10.1158/0008-5472.CAN-21-4349

Figure Lengend Snippet: SUMOylation promotes DDX39B-mediated circNCOR1 nuclear exportation. A and B, Representative images ( A ) and histogram ( B ) of circNCOR1 location in low- and high-grade bladder cancer tissues. Scale bars, 50 μm. H&E, hematoxylin and eosin. C and D, FISH ( C ) and subcellular fraction analysis ( D ) of circNCOR1 cellular localization in bladder cancer cells. Scale bars, 5 μm. E and F, Subcellular fraction analysis of circNCOR1 in UM-UC-3 ( E ) and UM-UC-1 ( F ) cells after overexpressing or downregulating DDX39B . G, Western blotting verified the PTM of DDX39B in bladder cancer cells. H, Western blotting of the PTM level of DDX39B after inhibitor treatment in UM-UC-3 cells. I, Subcellular fraction analysis of circNCOR1 cellular localization in indicated UM-UC-3 cells. J, Western blotting of the SUMOylation type of DDX39B in UM-UC-3 cells. K, Western blotting verified the SUMO2 modification of DDX39B after coimmunoprecipitation with anti-DDX39B and IgG control in UM-UC-3 cells. L, Schematic illustration of the SUMOylation sites on DDX39B predicted by GPS-SUMO. M, Sanger sequencing evaluation of the DDX39B K53R and DDX39B K155R mutations. N, Western blotting verified that the SUMO2 modification site of DDX39B was the K53 residue. O and P, FISH ( O ) and subcellular fraction analysis ( P ) of circNCOR1 in indicated UM-UC-3 cells. Scale bars, 5 μm. The statistical difference was assessed with the χ 2 test in B , E , F , I , and P . Error bars show the SD from three independent experiments. **, P < 0.01.

Article Snippet: LentiCRISPR v2 vectors containing single-guide RNAs targeting DDX39B were purchased from UBIgene and transfected into UM-UC-3 and T24 cells to construct the DDX39B knockout (KO) bladder cancer cells as described previously ( ).

Techniques: Western Blot, Modification, Control, Sequencing, Residue

Journal: Cancer Research

Article Title: Aberrant Nuclear Export of circNCOR1 Underlies SMAD7-Mediated Lymph Node Metastasis of Bladder Cancer

doi: 10.1158/0008-5472.CAN-21-4349

Figure Lengend Snippet: SUMOylated DDX39B mediates circNCOR1 nuclear exportation to promote bladder cancer LN metastasis. A – C, Representative images ( A ) and quantification of the tube formation ( B ) and Transwell migration ( C ) of HLECs cocultured with UM-UC-3 cells. Scale bars, 100 μmol/L. D – F, Representative bioluminescence images ( D ) and quantification ( E and F ) of popliteal LN metastasis in the nude mice ( n = 12 per group). G, The popliteal LN metastatic rate in the nude mice ( n = 12 per group). H and I, Representative IHC images ( H ) and percentages ( I ) of LYVE-1–indicated lymphatic vessel density in the footpad primary tumor tissues from the nude mice. Scale bars, 100 μm. The statistical difference was assessed by one-way ANOVA followed by Dunnett tests in B , C , E , F , and I , and the χ 2 test in G . Error bars show the SD from three independent experiments. **, P < 0.01. H&E, hematoxylin and eosin.

Article Snippet: LentiCRISPR v2 vectors containing single-guide RNAs targeting DDX39B were purchased from UBIgene and transfected into UM-UC-3 and T24 cells to construct the DDX39B knockout (KO) bladder cancer cells as described previously ( ).

Techniques: Migration

Journal: Cancer Research

Article Title: Aberrant Nuclear Export of circNCOR1 Underlies SMAD7-Mediated Lymph Node Metastasis of Bladder Cancer

doi: 10.1158/0008-5472.CAN-21-4349

Figure Lengend Snippet: circNCOR1 inhibits tumor growth in PDXs from LN metastatic bladder cancer. A, Timeline schematic for treatment of the mice carrying PDX. B and C, circNCOR1 and LY2157299 significantly inhibited PDXs growth in the mice ( n = 6 per group). D and E, qRT-PCR analysis of circNCOR1 ( D ) and SMAD7 ( E ) expression in PDXs before and after treatment ( n = 6 per group). F and G, qRT-PCR of DDX39B expression in bladder cancer (BCa) tissues versus NATs ( F ) and LN-positive versus LN-negative bladder cancer tissues ( n = 228; G ). H, Representative images of DDX39B and LYVE-1–indicated lymphatic vessel density in bladder cancer tissues. H&E, hematoxylin and eosin. Scale bar, 100 μm. I and J, Correlation analysis of DDX39B expression and LYVE-1–indicated lymphatic vessel density in bladder cancer tissues ( n = 228). The statistical difference was assessed by one-way ANOVA followed by Dunnett tests in B – E and the nonparametric Mann–Whitney U test in F and G . Error bars show the SD from three independent experiments. **, P < 0.01.

Article Snippet: LentiCRISPR v2 vectors containing single-guide RNAs targeting DDX39B were purchased from UBIgene and transfected into UM-UC-3 and T24 cells to construct the DDX39B knockout (KO) bladder cancer cells as described previously ( ).

Techniques: Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: Cancer Research

Article Title: Aberrant Nuclear Export of circNCOR1 Underlies SMAD7-Mediated Lymph Node Metastasis of Bladder Cancer

doi: 10.1158/0008-5472.CAN-21-4349

Figure Lengend Snippet: Clinical relevance of the DDX39B–circNCOR1–SMAD7 axis in patients with bladder cancer. A and B, circNCOR1 expression correlated positively with SMAD7 expression in the bladder cancer tissues ( n = 228). Scale bars, 50 μm. C, Correlation analysis of circNCOR1 and SMAD7 expression in bladder cancer tissues ( n = 228). D, SMAD7 expression levels were downregulated in bladder cancer tissues from The Cancer Genome Atlas (TCGA) database. E, qRT-PCR analysis of SMAD7 expression in bladder cancer (BCa) tissues and NATs ( n = 228). F, Comparison of SMAD7 expression in LN-positive and LN-negative bladder cancer tissues. G and H, Kaplan–Meier survival analysis of the OS ( G ) and DFS ( H ) of patients with bladder cancer with low versus high SMAD7 expression. The cutoff is the median. I, Schematic diagram describing that circNCOR1 activates SMAD7 transcription by recruiting hnRNPL to increase H3K9ac on SMAD7 promoter, thus inhibiting TGFβ–SMAD signaling pathway to suppress LN metastasis of bladder cancer, whereas SUMOylated of DDX39B promotes the nuclear exportation of circNCOR1 to impair the inhibitory role of circNCOR1 on bladder cancer LN metastasis. The statistical difference was assessed by the χ 2 test in B and the nonparametric Mann–Whitney U test in D–H . Error bars show the SD from three independent experiments. **, P < 0.01.

Article Snippet: LentiCRISPR v2 vectors containing single-guide RNAs targeting DDX39B were purchased from UBIgene and transfected into UM-UC-3 and T24 cells to construct the DDX39B knockout (KO) bladder cancer cells as described previously ( ).

Techniques: Expressing, Quantitative RT-PCR, Comparison, MANN-WHITNEY