?(Fig.3A).3A). of miR-34a, a known AR-targeting miRNA, contributes AR expression by XRN1. Thus we revealed the AR-miR-204-XRN1-miR-34a positive opinions loop and a dual function of miR-204/XRN1 axis in prostate malignancy. androgen-deprivation [23], in which LNCaP cells undergo NED [31]. Therefore CL1 cells might be a NEPC cell collection, due to its high expression of CD44 [32], a feature of PCa cells with NE phenotype [8-10]. In addition, we previously performed a comprehensive expression profiling analysis of LNCaP and CL1 cells using the massive parallel signature sequencing (MPSS) technology [33], and recognized 2088 MPSS signatures that are differentially expressed significantly (P<0.001) as listed in the Supplementary Table 2 of the publication [33]. Recently, Beltran et al. identified 1035 genes that are differentially expressed between NEPC and PCa tissues using RNA-seq [34]. Based on above information, we further compared the differentially expressed list of CL1 and LNCaP with the differentially expressed list of NEPC and CaP tissues, and identified an overlap of 35 genes. Among these 35 genes, 28 (80%, 28/35) of them changed in the same direction in the comparison (Supplementary Table 3), suggesting that CL1 and NEPC are more similar to each other than LNCaP and NEPC. Moreover, LASS2 antibody we detected a much higher expression of NSE and CgA, the two NE markers, in CL1 cells compared with that in LNCaP cells (Fig. ?(Fig.1D).1D). All these support that CL1 represents a NEPC subclone of LNCaP cells. Finally, our results further showed that miR-204 expression was significantly higher in the two NEPC cell lines (i.e. PC-3 and CL1) than that in the two PAC cell lines (i.e. LNCaP and 22Rv1) (Fig. ?(Fig.1E).1E). Taken together, our results Fagomine strongly suggest that AR is the key suppressor of miR-204 expression in PCa cells. miR-204 exhibits a dual regulation on PCa growth both and observation (Fig. 2A-C), our results showed that overexpression of miR-204 in 22Rv1 tumor cells resulted in a time-dependent reduction of tumor volume (Fig. ?(Fig.2E).2E). By contrast, overexpression of miR-204 significantly promoted the growth of CL1 (Fig. ?(Fig.2F)2F) Fagomine and PC-3 (Fig. ?(Fig.2G)2G) tumors in nude mice. These results again demonstrated the dual yet opposite role of miR-204 in regulation of tumor growth of Fagomine PAC and NEPC the algorithms of TargetScan 5.2 (http://targetscan.org/), PicTar (http://pictar.mdc-berlin.de/), and DIANA-microT v3.0 (http://diana.cslab.ece.ntua.gr/microT/). To validate Fagomine it, a luciferase reporter construct was generated by cloning a 562-bp-long 3-UTR of XRN1 mRNA downstream of the Renilla luciferase gene. Subsequently, our assay indicated that the luciferase activity in this reporter was inhibited by 47.8% in LNCaP cells (Fig. ?(Fig.3A).3A). Futhermore, the mutations introduced to the miR-204-pairing sequence in 3-UTR of XRN1 almost reversed the inhibition of luciferase activity by miR-204 (Fig. ?(Fig.3A),3A), indicating that miR-204 directly targeted the 3-UTR of XRN1. In support of this, ectopic expression of miR-204 lowered the level of XRN1 protein in all the PCa cell lines tested (Fig. ?(Fig.3B),3B), whereas introduction of the miR-204 inhibitor raised level of XRN1mRNA (Fig. ?(Fig.3C),3C), suggesting that miR-204 is a repressor of XRN1 expression in PCa cells. Open in a separate window Figure 3 XRN1, as a miR-204 target, is a dual regulator of PCa cell growth(A) Luciferase assay of the reporter gene with wild-type (WT) or mutant (MU) 3-UTR of XRN1 in LNCaP cells infected with or without miR-204-expressing lentivirus. (B) Western blot analysis of XRN1 expression in PCa cells in the presence of ectopic expression of miR-204 as indicated. (C) Levels of XRN1 mRNA in LNCaP and CL1 cells transfected with the miR-204 inhibitor or control oligonucleotides. (D) Western blot analysis of XRN1 expression in PAC cells with knockdown of AR. LNCaP and 22Rv1 cells were transfected with AR-siRNA or control RNA duplex. (E) Western blot analysis of the effect of miR-204 overexpression on regulation of XRN1 expression by Fagomine androgen in LNCaP cells. (F) Western blot analysis of XRN1 in PCa cells transfected with XRN1 siRNA. (G and H) Effect of silencing of XRN1 on cell growth (G) and clonogenicity (H) of PCa cells. The data were obtained from at least three independent experiments, and the values are shown as the mean SEM; * p< 0.05; **p<0.01. Given that miR-204 suppressed XRN1 expression (Fig. ?(Fig.3B),3B), it suggests that AR might up-regulate XRN1 expression its inhibitory effect on miR-204 expression (Fig. ?(Fig.1B).1B). Consistent with this, introduction of AR-siRNA decreased XRN1 expression in LNCaP and 22Rv1.