In the present work, we demonstrate that the expression of Ccnb1 depends on components of miRNA biogenesis (Drosha and Dicer) and function (Ago1 and Ago2) in mouse cells. We identify several miRNAs (e.g. miR-744, miR-1186, etc.) with predicted target sites in the Ccnb1 promoter that induce Ccnb1 expression. Mechanistically, we indicate that Ago1 and Ago2 are abundant in the nucleus consistent with previous observations (46
). Ago1 is localized to the proximal promoter region of Ccnb1 and miR-744 promotes RNAP II and H3K4me3 enrichment at the Ccnb1 TSS. Phenotypically, Ccnb1 overexpression has been reported to promote tumorigenesis and function as a putative oncogene in a variety of cancers (42
). As such, short-term overexpression of miR-744 or miR-1186 led to enhanced tumor cell proliferation in a manner similar to vector-mediated overexpression of Ccnb1. However, long-term expression in TRAMP C1 mouse cells altered chromosome composition suggesting prolonged Ccnb1 activation may cause chromosomal instability. Xenografts derived from early passage cells overexpressing miR-744 also possessed enhanced tumorigenicity, while cells from late passage populations displayed poor tumor growth in vivo
. Our data suggests that short-term expression of miR-744 and/or miR-1186 may promote in vivo
tumor growth through Ccnb1 activation; however, prolonged activation of Ccnb1 may cause chromosomal instability and inhibit tumor growth. This study reveals a physiologically relevant role for miRNA-mediated gene activation, which may be implicated in cancer initiation and/or development.
miRNAs are known to participate in a wide variety of cellular functions by suppressing gene expression via binding complementary sites in the 3′-UTR of mRNA sequences. Target predication in gene transcripts is largely dependent on sequence complementarity within the ‘seed’ region and target conservation across multiple species (50
). However, without experimental validation, such computational predictions are not always bona fide
). Predicting miRNA target sites in gene promoters is even more challenging due to a lack of relevant experimental data and general poor conservation of promoters across different species. However, by repurposing target prediction algorithms (e.g. miRanda) to scan promoter sequence for sites complementary to miRNAs, numerous putative targets can be identified with reasonably high score (22
). As such, it has been postulated that miRNAs may also play a regulatory role in fine-tuning gene transcription in the nucleus (52
analysis utilizing the miRanda algorithm (35
) revealed several miRNAs with sites highly-complementary to sequence in the Ccnb1 promoter. Of the five miRNAs tested, three resulted in Ccnb1 induction. Additionally, we previously reported two artificial dsRNAs with perfect complementary to target sites located at −313 and −597 relative to the TSS that also activated Ccnb1 expression (18
). The ability to simply activate gene expression cannot exclude the possibility that miRNAs/dsRNAs are functioning through a canonical mechanism of gene silencing and Ccnb1 induction is a consequence of downregulation of other regulatory genes. If taken into consideration that each functioning miRNA and dsRNA possesses a different ‘seed’ sequence and targets non-overlapping regions, the likelihood that each of the five duplexes induces Ccnb1 expression via suppression of common or different upstream regulatory genes is potentially low. Their only apparent similarity is that they all putatively target the same gene promoter. ChIP analysis did reveal an enrichment of Ago1 at the Ccnb1 promoter in vicinity close to the putative miR-744 target site providing a direct linking between the miRNA machinery and Ccnb1 promoter. However, miRNAs failed to increase Ccnb1 promoter activity in two reporter systems including a stably integrated Ccnb1 promoter-GFP chimera and transiently transfected luciferase reporter. We reason that promoter environment plays a critical role in determining susceptibility to gene activation mediated by miRNA. The cloned promoter may not fully recapitulate context of the endogenous environment. For instance, chromatin state, nucleosome positioning and/or the presence of potential regulatory non-coding transcript(s) may not be preserved in the exogenous promoters. Models have implicated overlapping antisense and/or promoter-derived cryptic non-coding RNAs as target molecules for facilitating RNAa and TGS (8
). Such transcripts may derive from sites outside the cloned promoter (e.g. intragenic regions), which would be missing in the reporter constructs.
We and others have previously shown that RNAa mediated by synthetic dsRNA with perfect complementary to promoter sequence requires Ago2 for optimal activity (33
). However, gene knockdown and overexpression experiments performed within this study revealed Ago1 had greater impact on Ccnb1 expression than Ago2 in context to miRNA-mediated Ccnb1 induction. ChIP analysis further indicated a selective enrichment of only Ago1 protein at the Ccnb1 promoter. Mescalchin et al.
) has shown that Ago2 is primarily involved in siRNA-mediated silencing pathways, while Ago1 and other family members are primarily involved in miRNA-mediated gene regulation. This segregation in Ago protein function may also remain for mechanisms of transcriptional regulation mediated by miRNA or perfect dsRNA duplexes. In support, Ago1 has been reported to be involved in miRNA-mediated TGS (31
Prostate cancer progression is accompanied by accumulating genetic alterations and CIN (58
). As such, features associated with karyotypic instability have been identified in human prostate cancer cell lines (59
). Similarly, the mouse prostate TRAMP C1 cell line also showed complex chromosomal abnormalities including polyploidy, aneuploidy and structural rearrangements indicative of chromosome/genome instability. It is noteworthy to point out that Ccnb1 overexpression appeared to enhance CIN in TRAMP C1 cells by promoting further numerical and structural alterations including loss of chromosomes (aneuploidy) and gain of additional marker chromosomes. Interestingly, miR-744 and miR-1186 also lead to similar chromosomal irregularities suggesting miRNAs (i.e. miR-744) might impair chromosome segregation/maintenance machinery via activation of Ccnb1 and render cancer cells more susceptible to cytogenetic alterations over time
It has been proposed that aneuploidy promotes tumorigenesis (45
). However, studies have also shown it may function as a tumor suppressor in context to some cells (61
). We made an unexpected finding in that high passage cells overexpressing miR-744 or miR-1186 possessed reduced tumorigenicity in the TRAMP C1 xenograft model. We speculate that the cytogenetic alteration resulting from Ccnb1 activation may have reduced viability and hampered TRAMP C1 cell growth. Cancer cells possess a dynamically balanced genome in order to maintain a growth advantage. The cytogenetic alteration caused by miRNA or Ccnb1 overexpression may disturb this balance resulting in a loss of some tumorigenic features.
In summary, the present study provides evidence that miRNA-mediated upregulation of gene expression functions in physiological context of cancer within a mouse model. We show activation of Ccnb1 by miRNA alters the growth potential of TRAMP C1 cells both in vitro and in vivo. Short-term overexpression of miR-744 or miR-1186 leads to enhanced cell proliferation, while long-term expression alters chromosome composition and negatively impacts tumor growth. We speculate that endogenous miRNA may facilitate RNAa in cancer to fine-tune gene expression and manipulate cancer cell phenotype. Further study is needed to identify additional examples and elucidate mechanism(s) by which miRNAs directly activate gene expression and oncogenic pathways.