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Supplementary Figure 4 from DDX31 Regulates the p53-HDM2 Pathway and rRNA Gene Transcription through Its Interaction with NPM1 in Renal Cell Carcinomas

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posted on 2023-03-30, 21:34 authored by Tomoya Fukawa, Masaya Ono, Taisuke Matsuo, Hisanori Uehara, Tsuneharu Miki, Yusuke Nakamura, Hiro-omi Kanayama, Toyomasa Katagiri

PDF file - 1.2MB, (A) FACS profiles of KMRC-1 (p53 wildtype) cells that were treated with siDDX31 or siEGFP. Cells were harvested with supernatant, stained with propidium iodide and analyzed by flow cytometry at 96 h after transfection. (B) Effects of DDX31 knockdown on cell growth and p53 and/or p21 expression in p53 mutant cell. Quantitative RT-PCR showing the suppression of endogenous DDX31 expression by DDX31 siRNA oligonucleotides (left). MTT assay showed a slight decrease in the cell numbers upon DDX31 knockdown in KMRC-20 (right ).(C) Effects of DDX31 knockdown on p53 and p21 protein levels in KMRC-20



Studies of renal cell carcinoma (RCC) have led to the development of new molecular-targeted drugs but its oncogenic origins remain poorly understood. Here, we report the identification and critical roles in renal carcinogenesis for DDX31, a novel nucleolar protein upregulated in the vast majority of human RCC. Immunohistochemical overexpression of DDX31 was an independent prognostic factor for patients with RCC. RNA interference (RNAi)-mediated attenuation of DDX31 in RCC cells significantly suppressed outgrowth, whereas ectopic DDX31 overexpression in human 293 kidney cells drove their proliferation. Endogenous DDX31 interacted and colocalized with nucleophosmin (NPM1) in the nucleoli of RCC cells, and attenuation of DDX31 or NPM1 expression decreased pre-ribosomal RNA biogenesis. Notably, in DDX31-attenuated cells, NPM1 was translocated from nucleoli to the nucleoplasm or cytoplasm where it bound to HDM2. As a result, HDM2 binding to p53 was reduced, causing p53 stablization with concomitant G1 phase cell-cycle arrest and apoptosis. Taken together, our findings define a mechanism through which control of the DDX31–NPM1 complex is likely to play critical roles in renal carcinogenesis. Cancer Res; 72(22); 5867–77. ©2012 AACR.

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