Renal cancer cells secrete exosomal ncRNAs that affect tumor microenvironment

Cancer cells secrete multiple molecules that affect tumor microenviroment. In our previous study we analyzed the secretory proteome and metabolome of renal cancer cells (PMID: 36604669). Small non-coding RNAs (sncRNAs) encompass a variable group of oligonucleotides, including miRNAs, piRNAs, and tRNA-derived fragment that can affect the expression of target genes. Here, we aimed to analyzed sncRNAs that are secreted by renal cancer cells and can affect tumor microenvironment. To this end, we: i) collected the conditioned media (CM) from two renal cancer-derived cell lines, Caki-1 and KIJ265T, as well as RPTEC, the control non-tumorous kidney cell line derived from proximal tubules; ii) used the CM for isolation of exosomal total RNA; iii) performed RNAseq to target and analyze the profiles of sncRNAs. We identified multiple miRNAs, piRNAs and tRNAs that were differently secreted by renal cancer cells when compared with non-tumorous proximal tubules. The analysis was followed by qPCR validation and functional analysis to reveal sncRNAs that affect tumor microenvironment. Caki-1 and RPTEC/TERT1 cells were cultured in accordance with manufacturer’s protocol (ATCC). KIJ265T were cultured as previously described (Boguslawska J, Piekielko-Witkowska A, Wojcicka A, Kedzierska H, Poplawski P, Nauman A. Regulatory feedback loop between T3 and microRNAs in renal cancer. Mol Cell Endocrinol. 2014;384(1–2):61–70). For the collection of conditioned media (CM), 10^6 cells were seeded at 75cm2 flasks, cultured for 24h, rinsed once with PBS and four times with DMEM (low glucose, no glutamine, no phenol red) (Gibco/Thermo Fisher Scientific, Paisley, UK) with GLUTAMAX (Gibco/Thermo Fisher Scientific, Paisley, UK). Following the addition of 15ml of DMEM (no phenol red) supplemented with GLUTAMAX, the cells were cultured for another 24h. CM were collected (three independently cultured batches per each cell line), centrifuged, aliquoted, and stored at -80oC. Exosomes were purified from CM, with the following isolation of total exosomal RNA. RNAseq was performed using Illumina NextSeq 500 sequencing platform.