Identification of miR-205 targets using an RIP-Chip assay with AGO2 antibody. Homo sapiens

In this study, the prognostic properties of miR-205 expression levels are investigated in a well-documented prostate cancer cohort. We show that miR-205 is correlated to shortened overall survival, significantly dividing the PCa patients into high and low risk groups. Furthermore, miR-205 is shown to inversely correlate to occurrence of metastases. In situ hybridization is also performed, demonstrating high miR-205 expression in the basal cells of benign prostate tissue glands. A RIP-Chip assay using an AGO2 antibody was implemented and the miR-205 targets identified were found to be enriched in MAPK/ERK, Toll-like receptor and IL-6 signaling pathways. We also found individual targets involved in cancer and androgen receptor signaling. Ectopic levels of miR-205 are shown to decrease the level of androgen receptor both at the mRNA and protein levels in prostate cancer cell lines. This is further corroborated in the prostate cancer cohort were miR-205 expression levels in the prostatic tissues are found to inversely correlate to assessment of androgen receptor (AR) immunostaining and to serum levels of PSA, a protein regulated by AR signaling. The level of miR-205 is also found to be significantly lower in castration resistant prostate cancer patients than in hormone naïve patients. Our data indicates that miR-205 is regulated by androgens and act by different mechanisms in androgen depleted settings, e.g. giving opposite effects on adhesion. Taken together these findings imply that miR-205 might have therapeutic potential especially for the castration resistant and currently untreatable form of prostate cancer. Overall design: Experiment done with biological triplicates. Three with miR-205 ectopic expression and three with negative control mimic ectopic expression. Followed by a RIP-Chip, ending with mRNA extraction and gene expression array.

本研究针对一组记录完备的前列腺癌队列，探究了miR-205（微小RNA-205）表达水平的预后价值。研究结果显示，miR-205与总生存期缩短显著相关，可将前列腺癌（PCa）患者清晰划分为高风险与低风险两组。此外，miR-205与肿瘤转移的发生呈负相关。本研究同时开展了原位杂交实验，证实良性前列腺组织腺体的基底细胞中miR-205呈高表达状态。我们采用AGO2抗体开展了RNA免疫沉淀芯片（RIP-Chip）实验，鉴定得到的miR-205靶基因显著富集于MAPK/ERK、Toll样受体以及IL-6信号通路中。此外还鉴定出参与癌症发生及雄激素受体（androgen receptor, AR）信号通路的单个靶基因。实验证实，在前列腺癌细胞系中，过表达miR-205可降低雄激素受体在mRNA及蛋白水平的表达量。这一结果在前列腺癌队列中得到进一步验证：前列腺组织中miR-205的表达水平与雄激素受体（AR）免疫染色评分以及AR信号通路调控的血清前列腺特异性抗原（PSA）水平均呈负相关。去势抵抗性前列腺癌（castration resistant prostate cancer, CRPC）患者的miR-205表达水平显著低于激素初治患者。本研究数据表明，miR-205受雄激素调控，并在雄激素剥夺环境下通过不同机制发挥生物学功能，例如对细胞黏附产生相反的调控效果。综上，上述研究结果提示miR-205可能具有治疗潜力，尤其针对当前尚无有效治疗手段的去势抵抗性前列腺癌。整体实验设计：实验设置生物学重复三组，分别为miR-205过表达组与阴性对照模拟物过表达组各三组。随后开展RNA免疫沉淀芯片（RIP-Chip）实验，最后提取mRNA并进行基因表达芯片检测。