The transcriptional regulation of miR-21, its multiple transcripts, and their implication in prostate cancer

Abstract

MicroRNAs (miRNAs) are a natural part of the most recently discovered and global regulatory pathway known as RNA interference. Functional studies have shown how specific miRNAs can function as tumor suppressors or oncogenes and, correspondingly, deregulated miRNA profiles have been observed in prostate and other cancers. However, the upstream pathways which regulate miRNA expression are only currently being uncovered. The Androgen Receptor (AR) is a nuclear hormone receptor and transcription factor which plays a paramount role in prostate cancer (PCa) pathobiology. We performed high throughput miRNA microarray analysis on two AR-responsive cell lines to identified 16 candidate AR-regulated miRNAs.(1) One of the most androgen-induced candidates was a known oncogenic miRNA, miR-21. In a small study of early grade PCa samples we found that miR-21 levels were frequently elevated in comparison to adjacent normal tissue. This observation was supported in the literature(2,3) and suggests clinical relevance. We found that the activated AR directly interacts with miR-21 regulatory regions, indicating direct transcriptional induction. Furthermore, we provide new reporter studies supporting AR-regulation. Importantly, in functional studies, we found that a modest overexpression of miR-21 enhanced tumor xenograft growth and was sufficient to support androgen-independent proliferation following surgical castration. Thus, our studies suggest a model where miR-21 contributes to androgen-dependent and androgen-independent PCa growth. However, the AR is only one of many reported transcriptional regulators of miR-21. Here we review our recent discoveries and further analyze the reported miR-21 regulatory regions, inhibitory and stimulatory signaling pathways, and primary transcripts.

Figure 1

MiR-21 mediated pathways. Several signaling pathways, including AR-mediated, stimulate miR-21 expression. Elevated miR-21 levels contribute to androgen-dependent proliferation and are able to fully support castration resistance. Those pathways in red indicate novel pathways which we recently identi3ed PCa models. Pathways in black refer to the previously reported pathways described in other models.

Figure 2

Genomic organization and transcripts of miR-21 gene Locus. The miR-21 hairpin is located on chromosome 17q23.1 immediately downstream of the coding gene, TMEM49. While TMEM49 and miR-21 transcripts overlap, they are independently regulated. White boxes represent terminal TMEM49 exons 11 and 12. Three previously identi3ed and functional promoter regions for pri-miR-21 are represented at the bottom of the 3gure. Promoter elements and positive (blue) and negative (red) transcriptional regulators are mapped above. Transcription Start Sites (TSS) are indicated by arrows. TMEM49 and miR-21 poly(A) signals are indicated by A.

Figure 3

miPPR-21 promoter activity in PCa. We reproduced the strategy reported by Fujita et al. to clone miPPR-21 in a PGL3 basic vector (InVitro-gen) further modi3ed to accommodate an Internal Ribosome Entry Site (IRES) upstream the Luciferase ORF. C4-2 cells were plated in 96 well plates 24 hours prior to Lipofectamine 2000 transfection with the indicated vectors and Renilla luciferase control. Fire3y luciferase activity was measured and normalized to a Renilla luciferase activity after lysing the cells. Results are expressed as Luciferase arbitrary units (a.u). The data represent 3 independent measurements. (A) PMA induction. Transfected C4-2 cells were challenged for 5 hours with vehicle (DMSO, white columns) or 2 nM PMA (black columns). (B) AR Inhibition. Transfected C4-2 were maintained for 48 hours in RPMI complete media plus 10% FBS either in combination with Vehicle (DMSO, white columns) or 10 μM of AR-antagonist (Casodex, black columns) prior to lysis.