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Previous Article | Next Article 
Molecular and Cellular Biology, July 2008, p. 4609-4619, Vol. 28, No. 14
0270-7306/08/$08.00+0 doi:10.1128/MCB.01652-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Department of Medicine, Department of Veterans Affairs and University of California, San Francisco, California,1 Program of Development and Stem Cell Biology, Department of Medicine, University of California, San Francisco, California2
Received 6 September 2007/ Returned for modification 17 October 2007/ Accepted 30 April 2008
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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Regulation of HOXA9 protein or mRNA levels by microRNAs (miRNAs) has not been reported. The programs available at the inception of this project (23, 24, 28, 35) gave various predictions for putative miRNA target sites within the 
1,160-nt 3' untranslated region (UTR) of the major Hoxa9 transcript (GenBank accession number BC006537). However, we were particularly intrigued by the fortuitous observation that one program utilized an alternatively spliced variant that designates the homeobox as the 3' UTR to predict that miRNAs would target the Hoxa9 homeobox region. Since previous studies had shown that several mammalian Hox genes could partially functionally replace their fly homologues in situations where essentially only the homeobox and an EXD-PBX interaction motif are conserved (36, 37), much of the biological activity of HOX proteins appears to reside within the homeodomain, with little biological activity other than the PBX interaction motif (4) within the flanking regions. Thus, the prediction that miRNAs would target the homeobox region appeared to be an especially appealing area for investigation. Since the defining feature of the Hox genes is their highly conserved homeoboxes, part of the unexplained function of these genes is their differential activity when they are coexpressed in highly overlapping patterns (30). The elucidation of possible regulation of these genes by miRNAs, which are thought to exquisitely sense single nucleotide differences, might provide a new level of control of these master regulatory genes.
In the current study, Hoxa9-immortalized bone marrow (BM) cells were used in conjunction with retroviral vector-mediated expression of hairpin miRNA precursors in proof-of-principle studies which showed that miR-126, miR-145, and let-7a target their predicted sites within the Hoxa9 homeobox. The miRNAs downregulated HOXA9 protein and its associated biological activity. Sequence homology analysis demonstrated that the putative miRNA binding sites were constrained beyond the level of constraint required for maintenance of protein coding, suggesting additional functionality. Quantitative reverse transcription-PCR (QRT-PCR) of hematopoietic progenitor cells showed that miR-126 and Hoxa9 are expressed in parallel in HSCs but are inversely expressed in myeloid leukemic lines and primary leukemia BM samples, suggesting that variation in levels of miR-126 might regulate HOXA9. This prediction was supported by the findings that the forced expression of miR-126 downregulated endogenous HOXA9 in MLL-ENL cells while the inhibition of endogenous miR-126 upregulated HOXA9 protein in F9 cells. We hypothesize that miR-126, in conjunction with the ubiquitous let-7s, normally targets Hoxa9 during early HSC differentiation but that in myeloid leukemias, the loss of this regulatory miRNA might elevate the HOXA9 oncoprotein.
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MATERIALS AND METHODS |
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Retrovirus-mediated expression of miRNAs.
Murine genomic regions containing the miRNA hairpins and 150 nt of 5' and 3' flanking regions were cloned into the MDH vector (5). High-titer retroviruses were produced by transient transfection of 293T cells and viral supernatants collected at 48 and 72 h posttransfection.
Cell transfection, BM transduction and immortalization, and Western blotting. Immortalized lines were produced by transducing 5'-fluorouracil (5'-FU)-treated BM progenitors with a truncated Hoxa9 cDNA lacking the 3' UTR, the same cDNA with mutated binding sites for the three miRNAs in the homeobox (mutHoxa9), or a modified full-length Hoxa9 cDNA, each cloned into an MSCV vector containing a neomycin selection cassette (12). F9 cells seeded at 0.4 x 106 in 1 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS) were cultured at 37°C for 4 h, transfected with 4 µl of DharmaFECT-1 and 50, 100, or 200 nM of miRNA-126-3p inhibitor (I-310017-02) or miRIDIAN miRNA inhibitor negative control 1 (IN-001000-01) (Dharmacon Inc., http://www.dharmacon.com), and assayed after 72 h. Proteins were assayed by Western blotting with antisera against HOXA9 (Upstate catalog no. 07-178), followed by antiserum to tubulin (Santa Cruz Biotechnology catalog no. 5286) or actin (Sigma catalog no. A5316) (12).
Fluorescence-activated cell sorting (FACS) and RNA analysis. Green fluorescence protein-positive (GFP+) transduced cells were sorted prior to Western blotting, colony assays, and growth (12). BM progenitors were sorted into long-term HSCs, short-term HSCs, and early progenitor pools in lysis buffer (Ambion catalog no. 1931) for RNA isolation through multichannel sorting (13). After approval by the UCSF Committee on Human Research, unidentified leukemia BM cells were placed in cell lysis buffer for RNA isolation. QRT-PCR was used to measure miR-126 and miR-145 or U6 RNA, by using Applied Biosystems kits (Applied Biosystems), and Hoxa9 or actin mRNAs (Table 1). Human leukemic cell lines were grown (34) and assayed for miRNA expression by Northern analysis, using antisense probes (Table 1). The Hoxa9 values were normalized to actin results and miR-126 values were normalized to U6 RNA results. To compare miR-126 expression levels between human and murine cells, miR-126 and U6 were measured in DAMI cells by QRT-PCR. miR-126 was measured by Northern analysis in all four human lines, and Hoxa9 values normalized to DAMI results are reported in Table 4.
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Statistical analysis. Statistical significance was calculated using the Student t test. All experiments were performed in duplicate, except where additional experiments were performed as noted.
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RESULTS |
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2.0-kb full-length Hoxa9 cDNA does not contain target seed sites for miR-126 or miR-145, while a single let-7 site is located near one of the polyadenylation signals in the 3' UTR, suggesting that the three miRNAs would target the homeobox region.
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8 or 4 times higher, respectively, than that observed for the triple vector (data not shown).
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90% or greater, the triple-vector efficiency was 40 to 50%. In order to compare knockdown efficiencies, cells were normally sorted for GFP+ expression prior to protein analysis. In replicate transduction experiments, the single miRNAs showed activity against HOXA9, with the triple vector exhibiting somewhat variable but normally the greatest amount of knockdown, with a mean decrease of 0.57% ± 0.23% (n = 10) (Fig. 2C). A portion of the observed variability may have been due to differences in miRNA expression levels, as reflected by significant differences in GFP expression between experiments. In all experiments, a nonspecific miRNA hairpin was included to control for possible effects on Dicer and Drosha caused by the exogenous hairpin RNAs. The miRNAs target the predicted sequences within the Hoxa9 homeobox. A mutant form of the Hoxa9 cDNA, in which the putative miRNA targets were altered at the nucleotide level while maintaining the wild-type amino acid sequence (mHoxa9) (Fig. 2D), was produced. This cDNA was used to produce an mHoxa9-immortalized BM cell line, in which the HOXA9 protein was expressed at levels similar to that expressed by the wild-type cDNA. Transduction of these cells with the triple-miRNA vector produced an insignificant decrease in HOXA9 protein (0.17% ± 0.17%, n = 4) (Fig. 2E). The effect of the triple vector on the wild-type HOXA9 protein (Fig. 2C) was significantly different from the decrease observed with the mutant protein (P = 0.01). These data indicate that the miRNAs are targeting the predicted sequences within the Hoxa9 homeobox.
miR-126, miR-145, and let-7 target the full-length Hoxa9 cDNA.
For historical reasons, all previous studies on HOXA9 immortalizing and leukemogenic activity (2, 12, 29) have used the truncated cDNA lacking 1,160 nt of the 3' UTR that we used for the initial studies of sensitivity to miRNAs directed against Hoxa9 described above. It was formally possible that the full-length endogenous Hoxa9 cDNA might be folded in such a manner as to be resistant to the miRNAs. Since initial experiments using a wild-type 3' UTR revealed that the putative polyadenylation signals were incompatible with efficient HOXA9 protein expression, we immortalized BM cells with a modified version of the full-length cDNA in which two polyadenylation signals had been removed. Transduction of this line with the triple miRNA knocked down the HOXA9 protein (Fig. 2F), showing that the targeting miRNAs could bind to a full-length Hoxa9 transcript (see also experiments described below using MLL-ENL cells).
miRNAs targeting the Hoxa9 homeobox block HOXA9 biological function. HOXA9-immortalized cells possess the capacity to form colonies following serial replating in semisolid media. Small-interfering-RNA-mediated loss of HOXA9 protein in these cells results in the loss of colony-forming activity (D. Garcia and C. Largman, unpublished data). Following FACS for GFP+, BM cells immortalized with the Hoxa9 lacking the 3' UTR and transduced with single, double, or triple miRNAs directed against Hoxa9 showed greatly diminished colony-forming capacity (Fig. 3A, left). In contrast, cells immortalized with the mutant HOXA9 protein formed colonies in the presence of the triple-miRNA vector (Fig. 3A, right). Similar experiments were performed to confirm that the combination of the three miRNAs reduces the biological activity of the full-length Hoxa9 cDNA (Fig. 3B). To confirm the capacity of the miRNAs to block HOXA9 biological activity, the growth of Hoxa9-immortalized cells in liquid culture was assessed. FACS-sorted cells transduced with either the single, double, or triple vectors expressing the miRNAs targeting HOXA9 showed substantially decreased growth rates compared to the control miRNA (Fig. 3C).
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miR-126 and Hoxa9 are inversely expressed in some primary myeloid leukemias.
To further explore the possible relationship between miR-126 and Hoxa9 expression, total RNA was isolated from BM samples from patients with acute promyelocytic leukemias. We noted that the samples with the highest Hoxa9 mRNA expression levels were low in miR-126, while samples with low Hoxa9 levels had some of the highest miR-126 levels (Table 4). Since, as discussed below, there are multiple pathways known to regulate Hoxa9 expression in leukemias, it is not surprising that there was not a statistically significant inverse correlation between Hoxa9 and miR-126 RNA levels. However, these data reenforce the cell line data that suggest a possible inverse relationship between miR-126 and Hoxa9 expression in leukemias. To assess whether the levels of miR-126 achieved by the viral vector were of similar magnitude to endogenous miR-126 expression, a series of human and murine cell lines were studied. As shown in Table 4, miR-126 expressed from the triple vector was of the same order of magnitude as that observed in human nonmyeloid cell lines and some murine lines. The viral-vector-driven expression was 50-fold higher than that measured in normal murine marrow or HSC progenitors but of similar magnitude to that detected in human erythro/megakaryocytic cells and several human leukemia samples.
miR-126 inhibits endogenous HOXA9 protein in MLL-ENL cells. To test the capability of miR-126 to regulate endogenous HOXA9 protein, we utilized a BM-derived cell line transformed with the MLL-ENL oncogene that had previously been shown to express high levels of endogenous HOXA9 (Fig. 5A, lane 1) (49). Introduction of miR-126 alone into MLL-ENL immortalized cells knocked down the endogenous HOXA9 protein, confirming that miR-126, probably together with endogenous let-7s, can target the endogenous full-length Hoxa9 transcript. It was not possible to replicate this finding in the well-studied myeloid leukemic lines that express high levels of Hoxa9 mRNA (34), because surprisingly, none of these cells express sufficient HOXA9 protein for experimental manipulation (for an example, see reference 48).
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95% of the F9 cells were transfected. Transient transfection with the miR-126 inhibitor resulted in increased HOXA9 protein relative to a control antisense RNA in a dose response fashion for the two higher doses used (Fig. 5B). Separate replicate experiments using the middle (100 nM) dose, recommended by the manufacturer, produced 1.9-fold and 1.6-fold increases in HOXA9 protein (data available on request). These data confirm that endogenous miR-126 can regulate endogenous HOXA9 protein levels. |
DISCUSSION |
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We believe the current report to be one of the first to show examples of miRNAs targeting the coding region of a mammalian transcript. The earlier literature on miRNAs suggested that most miRNAs target the 3' UTRs (39), a tendency that was reenforced by the fact that all of the earlier programs focused on the 3' UTRs for target prediction (23, 28, 35). This project, which focuses on the Hoxa9 homeobox region, was initiated following a serendipitous observation that one of these earlier programs treated a portion of the Hoxa9 coding region as part of the 3' UTR and predicted miRNA targets within the homeobox. Although there are substantial experimental data demonstrating that the 3' UTR can mediate translational repression (reviewed in reference 47), there is no a priori reason to exclude possible targeting to the coding regions. Indeed, manipulation of let-7 binding sites from the 3' UTR to the coding region did not prevent translational silencing in a model system (25). Although several programs that predict miRNA targets within the coding regions of mammalian genes have recently been described (20, 44), none of the programs predicted the three targeting miRNAs that we have discovered, illustrating the difficulty in making predictions given the limited seed requirements for miRNAs.
Much of the emphasis on the 3' UTRs apparently derived from the use of sequence conservation across species as a major predictor of miRNA targets, coupled with the presumption that conservation of functional coding regions would confound target identification. Indeed, the sequence conservation of the proposed miRNAs we identified might merely reflect the canonical conservation of HOX homeobox coding regions. However, given that first- and third-base codon changes can permit amino acid maintenance while allowing substantial nucleotide drift, the observation of very high nucleotide conservation of the miRNA target site nucleotides, accompanied by the expected variation at non-miRNA contact bases, across eight Hoxa9 homeoboxes (Fig. 1 and Table 2) implies functionality beyond conservation of amino acid identity. Thus, we propose that the miRNA targeting of the Hoxa9 homeobox is an ancient biological regulatory mechanism.
Grimson et al. (19) recently described an improved algorithm to predict miRNA targets based on five factors: (i) AU-rich composition near the target site, (ii) proximity to other miRNA sites; (iii) pairing to nt 13 to 16 within the miRNA, (iv) positioning within the 3' UTR relative to the stop codon, and (v) positioning away from the center of the 3' UTR. While parameters iv and v do not apply to the current miRNA targets, the other three conditions are at least partially met. The bias for AU-rich regions is consistent with a report of correlation between low 
G values for 70-nt flanking sequences and miRNA targets (50). Given that homeoboxes are GC rich (1), analysis of the G for 70-nt regions across the Hoxa9 cDNA did not identify the three sites within the homeobox compared to the overall sequence (C. Largman, unpublished data). However, the regions immediately around the three sites show that the miR-145 site is flanked by 7 of 10 and 6 of 9 A/Ts, while the 5' flanking regions of the let-7a and miR-126 sites contain 6 of 9 and 9 of 11 A/Ts, respectively. Thus, there are localized low-energy mRNA structures adjacent to the three Hoxa9 miRNA targets that may enhance complex formation. The let-7 site is 9 nt from the miR-145 binding site, while the binding site of miR-126 overlaps that of let-7 and is only 17 nt away from the miR-145 site. With regard to binding to sites 13 to 16 within the miRNAs, miR-126 forms a perfect match with the Hoxa9 sequence at nt 13 to 16; let-7a can form matches at nt 12, 13, 14, 17, 18, and 19; and in miR-126, nt 14 and 15 can bind to the Hoxa9 target.
We were able to readily demonstrate downregulation of either the retrovirally expressed or endogenous HOXA9 protein in blood cells by exogenous miR-126. However, the well-known inability of miR-126 to efficiently transfect hematopoietic cells precluded showing regulation by endogenous miR-126 in blood cells. We therefore used the nonhematopoietic F9 line as a suitable test system. Despite incomplete transfection and possible inefficient inhibition of the endogenous miR-126, F9 cells transfected with the inhibitor showed a mean 2.3-fold increase in HOXA9 protein, demonstrating that the endogenous protein is regulated by endogenous miR-126. Since there are no other putative miR-126 sites within the full-length Hoxa9 transcript, these data strongly suggest that miR-126 regulates endogenous HOXA9 protein by binding to the site we have identified within the homeobox.
While this work was in progress, two studies described analysis of miRNAs in hematopoietic progenitor cells (31, 40). Although Landgraf et al. reported qualitative detection of miR-126 in the CD34+ pool that contains HSCs (31), they did not examine expression in purified common-myeloid, megakaryocyte-erythrocyte, or granulocyte-monocyte early-myeloid-progenitor compartments. Monticelli et al. (40) did not report detection of either miR-126 or miR-145 in their samples. miR-126 has also been reported to be downregulated during terminal megakaryocytopoiesis but upregulated in megakaryocytic cell lines (16). Our data show that miR-126 expression parallels that of Hoxa9 mRNA in normal murine BM; both are relatively elevated in the HSC compartment and decrease during early-myeloid-progenitor cell differentiation. Low levels of miR-145 are present in all pools. These data suggest that miR-126 may keep HOXA9 levels in check during normal hematopoietic development. We reason that if miR-126 functions to block translation of Hoxa9 mRNA, then both RNAs should be present in the same cells. Unfortunately, the very low levels of HOXA9 protein expression and the low levels of HSC progenitors preclude analysis of whether the HOXA9 protein is altered by miR-126 in normal HSCs. In contrast to the parallel expressions of miR-126 and Hoxa9 in normal HSCs, there was a partial inverse expression pattern in leukemic cell lines and primary leukemia samples. Although this inverse relationship did not achieve a significant regression, this was not anticipated perhaps because there are numerous mechanisms reported to cause high-level Hoxa9 expression (3, 10, 41, 42, 49).
There is increasing evidence for the misregulation of miRNAs in cancer (38). It is of interest that human miR-126 is located on chromosome 9.34.3. Chromosome 9q34 appears to be a relative hot spot for chromosomal breakpoints associated with both myeloid and lymphoid leukemias (http://AtlasGeneticsOncology.org). The majority of leukemias associated with this region are chronic myelogenous leukemias arising from the t(9;22)(q34;q11) translocation which results in the Bcr-Abl fusion (reviewed in reference 9). In addition, there are a range of other leukemias that have been associated with the loss or rearrangement of 9q34. While 9q34 remains a relatively large region, it is unclear if breakpoints distal to the miR-126 locus can influence its expression. In this regard, a recent study showed that Hoxa9 was upregulated in BM samples of patients with chronic myelogenous leukemia arising from the Bcr-Abl fusion (9). There is now substantial data supporting a role for Hoxa9 in myeloid leukemias (reviewed in reference 22). Our data are consistent with a mechanism in which the loss of miR-126 expression would lead to increased levels of the HOXA9 protein through changes in mRNA and/or protein levels.
miRNA modulation of HOXA9 protein levels might confer an additional level of regulation to tissues in which the paralogous Hoxa9 genes are simultaneously expressed. Indeed, one of the long-recognized but perplexing aspects of Hox gene expression is that multiple members of a single paralogue, as well as those of closely related paralogues, are coexpressed in the same tissues during development. For instance, all of the Hox-10 and Hox-11 members are coexpressed in the developing skeleton, while three Hox-9 genes, including Hoxa9, are expressed in parallel in the developing mammary gland (6). Because all members of a paralogue would be expected to bind to the same DNA elements (45), such coexpression would potentially lead to redundant functions for these transcription factors. Almost all of the data defining Hox gene spatial patterning has been derived from in situ hybridization of mRNAs (30), with only a few examples of HOX protein expression analysis (26, 27). The finding that the miRNAs target the HOXA9 protein has implications for whether the protein products of other Hox genes actually parallel the previously defined mRNA expression patterns. One of the conclusions of the current study is that a reexamination of Hox gene expression at the protein level in various developmental programs may be warranted.
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ACKNOWLEDGMENTS |
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We thank R. Slany for the MLL-ENL cell line, C.-Z. Chen for the MDH vector, M. Loh for providing samples from the UCSF leukemic bone marrow cell bank, R. K. Humphries, M. McManus, and H. J. Lawrence for helpful discussions, and S. Fong for excellent technical support.
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FOOTNOTES |
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Published ahead of print on 12 May 2008.
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