Identification of novel AR-targeted microRNAs mediating androgen signalling through critical pathways to regulate cell viability in prostate cancer

Abstract

MicroRNAs (miRNAs) have been recognized as significantly involved in prostate cancer (PCa). Since androgen receptor (AR) plays a central role in PCa carcinogenesis and progression, it is imperative to systematically elucidate the causal association between AR and miRNAs, focusing on the molecular mechanisms by which miRNAs mediate AR signalling. In this study, we performed a series of time-course microarrays to observe the dynamic genome-wide expressions of mRNAs and miRNAs in parallel in hormone-sensitive prostate cancer LNCaP cells stimulated by androgen. Accordingly, we introduced Response Score to identify AR target miRNAs, as well as Modulation Score to identify miRNA target mRNAs. Based on theoretical identification and experimental validation, novel mechanisms addressing cell viability in PCa were unravelled for 3 miRNAs newly recognized as AR targets. (1) miR-19a is directly up-regulated by AR, and represses SUZ12, RAB13, SC4MOL, PSAP and ABCA1, respectively. (2) miR-27a is directly up-regulated by AR, and represses ABCA1 and PDS5B. (3) miR-133b is directly up-regulated by AR, and represses CDC2L5, PTPRK, RB1CC1, and CPNE3, respectively. Moreover, we found miR-133b is essential to PCa cell survival. Our study gives certain clues on miRNAs mediated AR signalling to cell viability by influencing critical pathways, especially by breaking through androgen's growth restriction effect on normal prostate tissue.

Figure 1. Flowchart of strategy.

This is the outline of the whole procedure for analysing microarray data to construct AR network in this study. Detailed steps are provided in the methodology and result sections.

Figure 2. Time discriminator for distinguishing early and late response stages.

Figure 2A. Time course profile of differentially expressed miRNA number. The dashed line refers to time discriminator for distinguishing early and late response stages. Figure 2B. Expression profile of miRNAs’ early- and late-response to DHT stimuli. Differential expression relative to 0 h is represented by . separates miRNA response into early and late stages. miRNAs are clustered into 4 groups: early upregulated (1), late upregulated (2), early downregulated (3) and late downregulated (4). Figure 2C. Venn diagram for number distribution of early responsive miRNAs and late responsive miRNAs. The red part denotes the androgen-resonsive miRNAs with response happened solely at the early stage, the blue part denotes miRNAs with response solely at the late stage, and the purple part denotes miRNAs with response both at the early and late stages. Figure 2D. Predicted ARE enrichment in early and late responsive miRNA genes. AREs are in the ±10 kb sequences flanking 5′-start site of pre-miRNAs.

Figure 3. Novel identification of AR target miRNAs.

Figure 3A. RS distribution of androgen-responsive miRNAs. miRNA numbers according to different RS values are presented, the dashed line denotes the threshold for identifying AR candidate primary target miRNAs. Figure 3B. RT-PCR analysis for identified AR candidate target miRNAs. Fold change of DHT-treated LNCaP cells over control samples was presented with significance assessment (in this study, *: p<0.05; **: p<0.01; ***: p<0.001). Fold change in control samples was deemed as 1 for all RT-PCR analyses in this study. This figure is the RT-PCR analysis of miRNAs at 40 min of DHT stimulation. Figure 3C. Schematic diagram of miR-133b, miR-19a, and miR-27a’s ARE location in the 5′ and 3′ regions. The horizontal arrows indicate the approximate ARE locations. Figure 3D. ChIP assay of AR-binding on candidate targets of miR133b, miR19a, miR27a. ‘IgG’ serves as the negative control for ChIP assay.

Figure 4. miR-19a's regulation on identified targets and prostate cancer cell viability.

Figure 4A. RT-PCR analysis of miR-19a’s identified significant targets: SUZ12, RAB13, SC4MOL, ABCA1 and PSAP. Fold change between miR-19a transfected samples and miR-NC (control) was presented with significance assessment. Figure 4B. Luciferase assays for miR-19a’s regulation on targets: SUZ12, RAB13, SC4MOL, ABCA1 and PSAP. Figure 4C–D. miR-19a’s contribution to prostate cancer cell viability. C. LNCaP cells cultured in androgen-depleted medium were treated with DHT, miR-19a and miR-NC separately, or in a combinational way. D. LNCaP cells cultured in androgen-depleted medium were treated with DHT, anti-miR-19a (si19a) and anti-miR-NC (siNC) separately, or in a combinational way. In both figures, the cell viability was measured by MTT assay during 4 days. MTT absorbance at each time point was presented.

Figure 5. Feedback regulation of AR expression and activity via miR-19a and PSAP.

Figure 5A–B. miR-19a’s influence on AR expression. miR-19a or miR–NC was transfected into the LNCaP cells for 24 h and 48 h, then AR’s mRNA expression (Fig. 5A) and the protein content (Fig. 5B) were observed. Figure 5C–D. miR-19a’s influence on AR activity. C. The nuclear protein content of AR after miR-19a or miR-NC transfection into LNCaP cells for 24 h and 48 h. D. AR’s nuclear protein content in LNCaP cells pre-transfected with miR-19a or miR-NC, then stimulated by DHT for 48 h. Figure 5E. miR-19a’s effect on the protein level of PSAP. The Western blotting result demonstrates miR-19a’s modest effect on suppressing PSAP.

Figure 6. miR-27a's regulation on identified targets and prostate cancer cell viability.

A. RT-PCR analysis of miR-27a’s identified significant targets: ABCA1 and PDS5B. B. Luciferase assays for miR-27a’s regulation on targets. C. The MTT assay for cell viability when transfected with DHT, miR-27a or miR-NC in four days. D. MTT assay for cells transfected with DHT, si-miR-27a or miR-NC.

Figure 7. miR-133b's regulation on identified targets and prostate cancer cell viability.

A. RT-PCR analysis of miR-133ba’s regulation on targets: CDC2L5, PTPRK, RB1CC1, and CPNE3. B. Luciferase assays for miR-133b’s binding on the targets. C, The MTT assay of LNCaP cells when transfected with DHT, miR-133b or miR-NC in four days. D. MTT assay for LNCaP cells transfected with DHT, si-miR-133b or miR-NC.

Figure 8. Global regulation of AR signaling by miRNAs.

.A. miRNA-mediated AR signalling network. B. miRNA dominant regulation. The Pie chart for miRNAs shows their dominant regulation on identified targets. 1, 2, 3, 4, 5, 6 and 8 represent the numbers of miRNAs, which co-regulate on the same target mRNA.