Use of prostate cancer cells as in vitro model for discovering miRNAs released by cells that developed docetaxel resistance

One of the major problems in androgen-independent prostate cancer (PCa), nowadays de ned castration resistant prostate cancer (CRPC), is the development of resistance to docetaxel (DCT). Considering the availability of new therapeutic agents it would be of great interest discovering predictive biomarkers of the early onset of a DCT resistance. In recent years circulating miRNAs have been validated as potential non-invasive biomarkers in several cancers, including PCa. So far, only few studies explored the connection between the modi cation of intra/ extracellular miRNAs and the development of a resistance to drug therapy. In this work we exploited PCa cells as in vitro model to identify miRNA involved in the resistance toDCT. Cells of the androgen-independent prostate cancer cell line DU-145 were treated with 3nM DCT. After 48h cells and culture medium were harvested and used to extract intra/extracellular RNA. The RNA was used to quantify extra/intracellular miRNA by qRT-PCR and by smallRNA-seq using TrueSeq smallRNA sample preparation kit and MiSeq sequencer (Illumina). DU-145 DCT resistant clones were isolated after about 30 days of continuous treatment with 3nM DCT. We detected miRNAs in cells and growth media before and after treatment of DU-145 with a concentration of DCT sub inhibitory for cell proliferation. The dot plot of the intra and extracellular miRNAs ratios showed that speci c miRNAs were differentially expressed/released by DU-145 DCT treated cells (DU/DCT-miRNAs). In addition, we showed that DU/DCT-miRNAs are speci c of DU- 145 cells as none of them were differentially expressed/released by PC-3, LNCaP and 22Rv1 prostate cancer cell lines treated with DCT. Finally, we report that DU-145 clones resistant to DCT differentially released 3 DU/DCT-miRNAs, suggesting that some of the miRNAs that change their release after an acute treatment may early indicate the presence of DCT resistant cells. Overall results indicate that the analysis of immediate post-treatment modifications may represent a useful strategy to discover miRNAs specifically released by PCa cells resistant to DCT. In perspective, these miRNAs may be candidates to be tested as predictive biomarkers of an early DCT resistance in CRPC patients candidate to DCT therapy.