Comparison of exosomal microRNAs secreted by 786-O clear cell renal carcinoma cells and HK-2 proximal tubule-derived cells in culture identifies microRNA-205 as a potential biomarker of clear cell renal carcinoma

摘要

Previous reports have indicated that the abundance of specific microRNAs (miRNA) contained within the exosome/microvesicle compartment of patient biofluids may be useful in diagnosing specific types of cancer. In the present study, the 786-O cell line, which is derived from a clear cell renal cell carcinoma (ccRCC), was used as an in vitro ccRCC tumor model and the human renal proximal tubule cell line HK-2 was used as its normal renal tissue control to investigate the similarities of exosomal content of selected ccRCC miRNA biomarkers in the supernatant with the content of those markers in the cells themselves. A PCR array identified miRNA biomarkers of solid RCC tumors (miR-210, MiR-34a, miR-155-5p and miR-150-5p) that were increased by 2–8 fold in 786-O exosomes compared with the control. These were subsequently chosen for further investigation using TaqMan RT-qPCR in addition to miR-15a and miR-205, which were selected based on prior interest as RCC biomarkers. MiR-15a, −34a, −210 and −155 levels were significantly lower in exosomes when compared with that in whole cells but did not differ between the HK-2 and 786-O cells in either the cytoplasmic, exosome or exosome-free supernatant fractions. By contrast, cytoplasmic miR-150 and miR-205 exhibited significant differences in concentration between the two cell lines. In addition, the cytoplasmic content of miR-150 and miR-205 was mirrored in the exosomal content of these miRNAs. Furthermore, the difference in exosomal miR-205 content was statistically significant. The present study indicated that measurements of the exosomal content of miR-205 and possibly miR-150, but not those of the other examined miRNAs, are proportional to their respective contents in the cells that secreted them. These findings suggest that in vitro RCC systems may be useful in identifying miRNAs with sufficiently high levels of exportation into exosomes; and with sufficiently different expression levels between tumor and normal cells to serve as ccRCC biomarkers in vivo.