In the study presented here, we first defined the in vitro
expression profiles of 157 human miRNAs in primary human LECs and BVECs using a TaqMan-based qRT-PCR profiling platform, whose increased sensitivity facilitated the detection of at least twice as many miRNAs in HUVECs as previously reported (19
). We also found that one of the most highly expressed HUVEC miRNAs, miR-126 (19
), was >600 times more abundant in both endothelial cell types than in either keratinocytes or fibroblasts. Comparative analysis identified four BVEC and two LEC signature miRNAs. Of the four BVEC signature miRNAs, three were previously reported as highly expressed in HUVECs (19
), and a very recent study has demonstrated that tumor necrosis factor treatment augments miR-31 expression in HUVECs (60
). Moreover, our miRNA profiling study has further classified the widely expressed (38
), metastasis-associated (63
) miRNA miR-31 as a BVEC signature miRNA. Finally, in agreement with their LEC-specific expression, neither miR-95 nor miR-326 was detected in the previous studies.
Importantly, further analysis of miR-31, miR-326, miR-125b, and miR-99a in adult mouse tissues confirmed that their vascular lineage-specific expression patterns were maintained in vivo
. The degrees of lineage-specific expression differences in vivo
were, however, usually less pronounced and more variable than those observed in vitro
. This is likely due to the mixed populations of BVECs and LECs isolated from the multiple vessel types present in the colon tissue (capillaries, postcapillary venules, lymphatic capillaries, lymphatic collecting vessels, etc.), which likely exhibit different gene expression patterns. Moreover, their relative contributions to the isolated total RNA might vary, thus contributing to larger variability in miRNA expression patterns. In addition, the ex vivo
miRNA expression profiling studies were technically challenging as the whole process took more than 2 h and only a few thousand endothelial cells could be isolated by high-speed cell sorting. Consequently, the smaller amounts of isolated total RNA, reduced RNA quality, and possible gene expression changes incurred during the 2-h isolation procedures likely contributed to the observed differences in in vivo
and in vitro
miR-31 expression, as well as to the observed interindividual variability in miR-31 expression. Surprisingly, we were unable to confirm the differential expression patterns of miR-137 in vivo
. This is likely because miR-137 expression levels were very low in the adult tissues analyzed here, as indicated by the late qRT-PCR detection (CT
, >35) and high standard deviations between technical replicates. ISH analysis of chicken embryos revealed that miR-137 is expressed in blood vessels and cardinal veins at stage 25 of embryonic development (8
), demonstrating that miR-137 expression is associated with the developing blood vasculature.
The identification of vascular lineage-specific miRNAs suggested that they might regulate fundamental and lineage-specific endothelial cell functions and/or differentiation processes. Indeed, overexpression of the BVEC-specific miRNA miR-31 in LECs induced the preferential degradation of LEC signature genes, including those for the well-characterized lymphatic transcription factors PROX1 and FOXC2. As these lymphatic lineage-specific molecules act as molecular switches, their preferential suppression suggests that BVEC-specific posttranscriptional regulatory mechanisms help maintain BVEC phenotypes by suppressing lymphatic lineage-specific transcription programs. This concept was supported by our findings that ectopic overexpression of miR-31 in LECs preferentially repressed LEC signature gene expression and induced BVEC signature gene expression. In this respect, our identification and validation of PROX1
as a direct miR-31 target are intriguing, as BVEC-specific posttranscriptional regulation of PROX1 could, at least in part, explain these in vitro
miR-31-mediated reprogramming events on the molecular level. Indeed, previous studies have demonstrated that PROX1
overexpression in BVECs induces the expression of lymphatic vascular markers and suppresses blood vascular markers (26
), whereas PROX1 knockdown in LECs inhibits LEC signature gene expression and triggers BVEC signature gene expression (44
; Shin et al., unpublished). Moreover, the overlaps between the miR-31-regulated genes identified here and a PROX1 loss-of-function data set further indicate that transcriptional reprogramming events observed following miR-31 overexpression in LECs were, in part, mediated by miR-31 repression of Prox1. Additional experiments are required to determine which of the miR-31-regulated candidate Prox1 target genes may also be direct targets of miR-31.
was not a predicted target gene of miR-31 (16
), our manual miR-31 site prediction analyses of the 5.4-kb PROX1
3′ UTR and subsequent luciferase 3′ UTR tethering assays identified a bona fide miR-31 binding site between nt 949 and 971 of the PROX1
3′ UTR. Interestingly, similar manual miR-31 prediction analyses of the chimpanzee, mouse, rat, chicken, Xenopus
, and zebrafish PROX1
3′ UTRs revealed that this site is evolutionarily conserved in vertebrates and identified additional, potentially functional, miR-31 binding sites (see Table S6 in the supplemental material). Taken together, our transcriptome profiling and biochemical studies have revealed a novel, highly conserved, BVEC-specific posttranscriptional regulatory mechanism that suppresses PROX1
expression in the blood vasculature.
Our findings also suggested that miR-31 expression in the developing blood vascular endothelium could regulate the acquisition of lymphatic lineage-specific characteristics and, thus, vascular development in vivo
. Multiple miR-31 loss-of-function studies using morpholino oligonucleotides were performed with both wild-type and plcg1
mutant zebrafish embryos. Statistically significant vascular phenotype differences were not observed in zebrafish embryos injected with low-to-moderate amounts (≤10 ng) of MO (data not shown). This suggests that the miR31-mediated regulation of vascular development identified here is redundant. This is not surprising, since miRNAs frequently function cooperatively (3
), which in turn complicates the attribution of specific functions to individual miRNAs (53
). In contrast, miRNA gain-of-function experiments have proven very informative and have defined important biological functions of several miRNAs (42
). For example, overexpression studies with Xenopus
embryos have demonstrated that miR-15 and miR-16 restrict the size of Spemann's organizer in vivo
by targeting the nodal type II receptor acrvr2a (42
). We therefore carried out miR-31 overexpression studies with Xenopus
and zebrafish embryos to determine the effect of miR-31 on cells and tissues that normally do not express miR-31, such as the lymphatic vasculature.
Our gain-of-function experiments clearly demonstrated that miR-31 expression is incompatible with normal lymphatic vascular development in Xenopus
and, to a lesser extent, zebrafish embryos. The analysis of Xenopus
embryos suggests that some aspects of lymphatic vascular development, such as specification of lymph hearts and LECs in the tail, are unaffected by miR-31 overexpression. Lymphangiogenesis and the development of an extensive lymphatic vasculature in the embryonic trunk are, however, clearly reduced and/or disrupted. Furthermore, we demonstrated that these observed lymphatic defects were reminiscent of those observed following MO-mediated inhibition of vegfc. These phenotypic similarities indicate that miR-31 overexpression interferes with an early step in lymphatic development. The identification of evolutionarily conserved miR-31 binding sites in PROX1
3′ UTRs (see Table S6 in the supplemental material) suggests that miR-31 overexpression may directly target and interfere with PROX1
transcripts in vivo
. Moreover, the abnormal or disrupted intersomitic vein sprouting seen in Xenopus
and zebrafish embryos (data not shown) following miR-31 overexpression implies that miR-31 also regulates BVEC responsiveness to the environmental stimuli directing blood vascular growth and maturation. Interestingly, several genes involved in the Slit/Robo, netrin, and ephrin signaling pathways (see Table S3 in the supplemental material), which provide crucial guidance cues during blood vascular development (1
), were repressed following miR-31 gain of function in vitro. In vivo
posttranscriptional regulation of any one of these molecules by miR-31 could contribute to the observed blood vascular maturation defects. Taken together, our results indicate that appropriate expression of miR-31 during vertebrate embryogenesis is required for both lymphatic vascular development and blood vascular growth and maturation. Interestingly, our in vivo
studies also correlate well with a recent study demonstrating that miR-31 controls the invasive capacity of breast cancer cells (63
). Collectively, these studies suggest roles for miR-31 in the regulation of cell migratory behavior during normal embryonic development and under pathological conditions in the adult body.
On the basis of our in vitro
studies, we postulate that PROX1 transcripts represent one of the key targets of miR-31. This repression would prevent inappropriate and/or premature transcriptional activation of lymphatic differentiation in the developing blood vasculature. While this notion is an attractive model, it is, however, important to stress that miR-31 targets several other LEC signature genes. It is therefore unlikely that posttranscriptional repression of PROX1
by miR-31 is solely responsible for the vascular developmental defects observed in Xenopus
and zebrafish embryos overexpressing miR-31. For example, miR-31-mediated repression of FOXC2, a transcription factor that is required for specification of the lymphatic capillaries versus collecting lymphatic vessels at later stages of embryogenesis (1
), may also contribute to the vascular defects observed. Another miR-31 candidate target is RAMP2, a calcitonin receptor-like receptor-associated receptor activity-modifying protein that triggers lymphangiogenesis in response to adrenomedullin signaling (13
). Finally, other LEC signature molecules subject to miR-31 regulation, whose lymphatic lineage-specific functions have not yet been characterized, could also enhance the effects miR-31 has on lymphatic and blood vascular development.
The miRNAs profiled in the present study represent approximately 25% of the known human miRNAs. Thus, more comprehensive and global miRNA profiling studies may result in the identification of additional endothelial lineage-specific miRNAs. In summary, we have defined the first vascular lineage-specific miRNAs and identified with miR-31 a novel miRNA-mediated regulatory mechanism that inhibits LEC phenotype acquisition in vitro and vascular development in vivo. From a therapeutic perspective, it remains to be investigated whether the ectopic expression of miR-31 might also inhibit malignant tumor-associated (lymph)angiogenesis, thus preventing tumor growth and cancer metastasis.