p27 regulates a variety of cellular processes, such as proliferation, differentiation, migration, and apoptosis, and its expression must be tightly controlled to prevent cell malfunction that could lead to tumor development. In this report, we have investigated the mechanisms that regulate the translation of p27 mRNA. Our results indicate that p27 mRNA is translated in a cap-dependent manner in HeLa and HL60 cells. In addition, we found that miR-181a inhibits cap-dependent translation of p27 mRNA in undifferentiated HL60 cells by virtue of two target sites in the 3′ UTR and that differentiation concurs with relief of miR-181a-mediated translational repression.
Several studies have reported the presence of an IRES in the 5′ UTR of p27 mRNA (8
). Our results, like those reported by Liu et al. (39
), contradict these findings. First, the use of promoterless DNA bicistronic constructs revealed the presence of cryptic promoters in the sequence encoding the p27 5′ UTR (Fig. ). The presence of mRNA species transcribed from these promoters was confirmed by RNase protection assays (data not shown), and their sizes match those transcription initiation sites described in previous studies (11
). Second, transfection of bicistronic mRNAs yielded no significant translation of the second cistron (Fig. ). Third, transfection of monocistronic mRNAs into HeLa or HL60 cells resulted in activity that was dramatically reduced by manipulation of the cap structure or insertion of a stable hairpin close to the cap, both treatments known to inhibit cap-dependent but not IRES-dependent translation (Fig. and ). Although we did not detect IRES activity in the p27 5′ UTR, we did observe a stimulatory role of this UTR in the context of cap-dependent translation (see below).
Many regulatory factors that control the translation of specific mRNAs operate through the 3′ UTR (13
). The importance of the 3′ UTR has been highlighted by the discovery of miRNAs and their essential role in the control of many cellular processes (reviewed in reference 6
). miRNAs have been shown to act as either tumor suppressors or oncogenes and are better predictors of tumor type and prognosis than mRNAs (36
). Recently, miRNAs have appeared as chief regulators of p27 mRNA translation. The first indications that miRNAs could play a role in the expression of p27 were obtained in Drosophila
). Depletion of Dicer-1, the RNase III enzyme required for miRNA biogenesis, delays G1
/S transition of germ line stem cells by increasing the amount of the p27 orthologue Dacapo. Subsequent studies have identified miR-221 and miR-222 as repressors of p27 synthesis in cell lines derived from glioblastoma, melanoma, hepatocarcinoma, papillary thyroid carcinomas, and prostate carcinoma (15
). Generally, inhibition of p27 expression by miR-221/miR-222 in these cells leads to increased proliferation and improved colony-forming potential. miR-221 is also expressed in HL60 cells, although it does not seem to control p27 expression in that cell line (Fig. ). Rather, we have found that another miRNA, miR-181a, represses p27 mRNA translation in undifferentiated HL60 cells (Fig. ). Furthermore, overexpression of miR-181a in THP1 cells leads to inhibition of endogenous p27 synthesis (Fig. ). miR-181a is preferentially expressed in hematopoietic tissues and cell lines, where it plays a critical role in the development of B and T cells through regulation of the expression of the key factors Bcl-2, CD69, and TCRα (7
). Regarding its function in myeloid differentiation, Debernardi et al. (12
) found a strong correlation between the levels of miR-181a and the expression of putative target mRNAs associated with a specific acute myeloid leukemia subtype. These studies showed that in acute myeloid leukemia samples of the M2 subtype, from which HL60 cells are derived, the amount of miR-181a is elevated and some of its putative targets are downregulated, whereas in the more differentiated samples of the M5 subtype, from which THP1 cells were obtained, low levels of miR-181a correlate with upregulation of the same targets. We have found a similar inverse correlation between miR-181a and p27 expression in HL60 and THP1 cells (Fig. ).
There are two target sites for miR-181a in the p27 3′ UTR that can function individually or synergistically to repress the translation of p27 reporters (Fig. ). The first site does not perfectly match the miR-181a seed region, but other features, including increased supplementary pairing, the presence of an adenine at position 1, and the AU-rich flanking regions, compensate for a suboptimal seed sequence (25
). This site coincides with one of the two functional miR-221 binding sites in the p27 3′ UTR. However, mutation of the two miR-221 sites had no effect on p27 mRNA translation, suggesting that miR-221 does not regulate p27 expression in HL60 cells (Fig. ). The distal site (positions 729 to 753) is located in a region for which we did not detect repressor activity in our initial 3′-UTR deletion analyses (Fig. , p27-F-[552-1341] mRNA). This discrepancy could be explained by the sequence context in which these mutations were analyzed, because further analyses using smaller fragments containing this site (p27-F-[552-870] mRNA) indeed revealed translational repressor activity (data not shown).
Repression by miR-181a is relieved during TPA treatment of HL60 cells, contributing to the high p27 levels necessary for full differentiation (Fig. to ). Other factors, such as regulatory RNA-binding proteins, could collaborate in the translational control of p27 mRNA at this time. The RNA-binding protein Dead-end 1 (Dnd1) interacts with U-rich sequences in the vicinity of the miR-221 target sites and blocks miR-221-dependent repression (32
). Therefore, it is conceivable that similar proteins could prevent the interaction of the residual miR-181a with its target sites during differentiation. In such cases, these proteins should bind to sequences different from those recognized by Dnd1, because mutation of these sequences does not affect translation of p27 in our model system (data not shown). The well-known regulators HuR and PTB could be good candidates, since both interact with p27 mRNA, albeit at the 5′ UTR, and have been shown to modulate p27 synthesis (8
). Interestingly, translational derepression during HL60 cell differentiation is hampered by the inversion of the p27 5′ UTR (Fig. ). Thus, although the effects of HuR and PTB were originally interpreted in the context of IRES-dependent translation, they could play roles in modulating the function of miR-181a. In support of this hypothesis, HuR performs a similar task in hepatocarcinoma cells under certain stress conditions, since it prevents the inhibition of the cationic amino acid transporter 1 (CAT-1) mRNA by miR-122 (4
Our results, as well as those of other laboratories, highlight the relevance of miRNA-mediated translational control of p27 mRNA. Although miRNAs usually repress translation about twofold, the observation that p27 is haploinsufficient for tumor suppression implies that a reduction of this kind would be enough to promote tumor growth. Learning about the regulation of p27 mRNA translation and transcript stability, two largely unexplored aspects of p27 biology, will expand the therapeutic approaches to fight against cancer.