Presentation Authors: Renpei Kato*, Morioka, Japan, Tomoya Fukawa, Tetsuro Yoshimaru, Yosuke Matsushita, Tokushima, Japan, Masaya Ono, Tokyo, Japan, Kei Daizumoto, Tokushima, Japan, Yoichiro Kato, Morioka, Japan, Toyomasa Katagiri, Tokushima, Japan, Wataru Obara, Morioka, Japan
Introduction: Renal cell carcinoma (RCC) is the most common malignancy of kidney, yet its molecular pathogenesis is poorly understood. We identified protein of relevant evolutionary and lymphoid interest domain containing 2 (PRELID2) as a novel clear cell RCC (ccRCC)-specific molecule through genome-wide expression profile analysis. To date, no reports have characterized the significance of transactivation of PRELID2 in clinical RCC progression. We here report the critical roles of a mitochondrial PRELID2 in renal carcinogenesis.
Methods: We used siRNA oligonucleotides to knock down gene expression in two ccRCC cell lines, Caki-1 and 786O. We established PRELID2 stably transfected HEK-293 cell line. . Cell viability was assessed by Cell Counting Kit-8 (DOJINDO) at 72hrs after transfection. The absorbance at 450 nm was measured using a multi-label counter infinite 200 (TECAN). Long-term cell proliferation was measured using by the IncuCyteÂ® ZOOM system (Essen BioScience). Mitochondrial reactive oxygen species (ROS) production measured by flow cytometry using MitoSOX Red (Thermo Fisher scientific). Mitochondrial respiratory function measured using Seahorse XF Cell Mito Stress Test XF24 extracellular flux analyzer (Agilent). We also analyzed PRELID2 functions by RNA interference and mass spectrometry, and evaluated PRELID2 interaction protein using immunoprecipitation with PRELID2 and mass spectrometry.
Results: DNA microarray, quantitative RT-PCR and TGCA database analyses showed significant upregulation of PRELID2 in ccRCC cases. Introduction of PRELID2 into HEK293 cells significantly enhanced cell growth, whereas knockdown of PRELID2 expression drastically suppressed growth in ccRCC cells. Notably, depletion of PRELID2 led to enhance ROS production, thereby causing increased oxidized proteins, in ccRCC cells. Extracellular flux analysis showed that downregulated of spare respiratory capacity in PRELID2-depleted cells. Furthermore, we found the interaction of HA-PRELID2 with endogenous PHB2 (Prohibitin 2), which is essential for mitochondrial morphogenesis and homeostasis, in ccRCC cells.
Conclusions: Our studies are the first to demonstrate that PRELID2 is likely to play a crucial role in renal carcinogenesis though its interacting with mitochondrial PHB2.