Identification of vascular lineage-specific miRNAs. qRT-PCR using individual TaqMan miRNA assays confirmed the lymphatic lineage-specific expression of miR-95 and miR-326 (A) and the BVEC-specific expression of miR-137, miR-31, miR-125b, and miR-99a (B). Data were normalized using RNU48 and are shown as mean relative abundances ± the standard deviations (n = 4/group). *, P ≤ 0.05; ***, P ≤ 0.01.

miR-31 overexpression in LECs modulates the expression of BVEC and LEC signature genes. (A and B) miR-31 was overexpressed in LECs via transfection with 2 μM pre-miR-31 precursor (n = 2) or pre-miR-Neg (n = 2). Microarray analysis after 48 h demonstrated that miR-31 overexpression repressed the expression of 33 LEC signature genes (A) and 23 BVEC signature genes (B). In addition, 4 LEC signature genes and 22 BVEC signature genes were induced. (C to E) TaqMan-based qRT-PCR analyses confirmed statistically significant miR-31-mediated repression of four of the LEC signature genes studied (C) and of two of the BVEC signature genes studied (D), as well as statistically significant induction of two of the BVEC signature genes studied (E). Data were normalized using β-actin and are shown as relative abundances ± the standard deviations. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Direct posttranscriptional regulation of PROX1 by miR-31. (A) Schematic representation of the full-length PROX1 3′ UTR and the consecutive fragments present in the PROX1 3′ UTR-luciferase reporter constructs (PROX1 FL to F6). Candidate miR-31 binding sites identified using standard nucleic acid alignment techniques (gray) and TargetScan 5.0 (black) are indicated. The PROX1-miR-31 base pairing is shown for the 7mer-m8 TargetScan-predicted binding site. (B to D) LECs or HUVECs were cotransfected with the plasmids containing a miR-31 binding site (miR31BS), the PROX1 3′ UTR (FL to F6), the PROX1 CDS, or PROX1 3′ UTR miR-31 binding site mutant forms (FL-mut or F2-mut) and the pre-miR-31 precursor, anti-miR-31 inhibitor, or negative-control molecules. (B) The activities of luciferase constructs containing miR31BS, PROX1 FL, and PROX1 F2 decreased significantly following miR-31 overexpression (pre-miR-31; n = 3) in LECs compared to those of negative controls (pre-miR-Neg; n = 3). (C) miR31BS and PROX1 FL reporter gene activities increased significantly following miR-31 inhibition (anti-miR-31; n = 3) in HUVECs compared to those of negative controls (anti-miR-Neg; n = 3). miR-31 loss of function also enhanced the activities of PROX1 F1 and PROX1 F2 constructs, but these differences were not statistically significant. (D) miR-31 mutant binding site full-length and F2 PROX1 3′ UTR (PROX1 FL-mut and PROX1 F2-mut) luciferase activities showed no major change following miR-31 overexpression (pre-miR-31, n = 3) or knockdown (anti-miR-31, n = 3). Data were normalized to firefly luciferase activities and are shown as mean relative abundances ± the standard errors. ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

miR-31 overexpression decreases venous sprouting in zebrafish embryos. (A) plcg1^t26480 mutant embryos in the TG(fli1a:gfp)^y1 background were injected with 340 pg of negative-control pre-miR-Neg (n = 71) or 340 (n = 46) or 170 (n = 24) pg of human pre-miR-31 precursor. Venous sprouts were quantified at 48 hpf. UIC, uninjected control (n = 120). Data are shown as averages ± standard deviations relative to the uninjected control of the same clutch. ***, P ≤ 0.001. (B to D) Lateral views of plcg1^t26480 mutant embryos in the TG(fli1a:gfp)^y1 background. Dorsal aorta formation and sprouting of intersegmental arteries are suppressed in mutant embryos, and therefore only venous sprouts (examples indicated by arrows) and parachordal lymphangioblasts (asterisks) are visible in the dorsal trunk. Examples of an uninjected control plcg1^t26480 mutant embryo (B) and plcg1^t26480 mutant embryos injected with 340 pg of negative-control pre-miR-Neg (C) or 340 pg of human pre-miR-31 precursor (D) at day 2 postfertilization are shown. PCV, posterior cardinal vein.

miR-31 is preferentially expressed by BVECs in vivo. (A) Mouse BVECs (CD45⁻ CD31⁺ podoplanin⁻) and LECs (CD45⁻ CD31⁺ podoplanin⁺) were isolated from adult mouse colons (n = 8) by FACS sorting. TaqMan qRT-PCR analysis of mmu-miR-31 demonstrated that miR-31 was at least 1.5-fold more abundant in BVECs from six of the eight mice. Data were normalized using sno234 and are shown as relative abundances ± the standard deviations. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, not statistically significant. (B to I) miR-31 (B, D, and F) and negative-control (G; sense miR-159) ISH and double-immunofluorescence analysis (C, E, H, and I) of serial sections of adult mouse colon tissues imaged at low magnification (×2.5; B to E) and high magnification (×20; F to I). Low magnification of ISH of adult mouse colon tissue revealed strong miR-31 staining in the majority of the cells (B and D), while double-immunofluorescence analysis of serial sections (C and E) and comparison of miR-31- and sense miR-159-probed sections at high magnification (F to I) demonstrated that miR-31 preferentially colocalized with blood vessels (CD31⁺ LYVE1⁻) present in the submucosa and mesenteric attachments of the colon (arrows), as well as the lamina propria (arrowheads). In contrast, lymphatic vessels (CD31⁺ LYVE1⁺) displayed weak or no miR-31 signals (asterisks). Panels F and G are high-magnification images of the boxed regions in panels D and E, respectively. Corresponding blood and lymphatic vessels in panels F to I are numbered. Scale bars are 200 μm (B to E) and 50 μm (F to I).

miR-31 gain and loss of function modulate PROX1 mRNA and protein levels. (A to C) Transfection of LECs with 4 μM pre-miR-31 precursor (n = 4) or pre-miR-Neg (n = 4). (A) qRT-PCR analysis revealed a ≥60% decrease in PROX1 transcripts after 48 h. (B) Immunoblotting of cell lysates with anti-PROX1 and anti-β-actin antibodies (loading control) demonstrated decreased PROX1 protein levels following miR-31 gain of function. The values to the left are molecular sizes in kilodaltons. (C) Semiquantitative analysis of PROX1 immunoblot signal intensities, relative to β-actin, defined a <40% reduction of PROX1 protein following miR-31 overexpression. Data are shown as relative abundances ± the standard deviations (n = 4/group). (D) qRT-PCR analysis showed that transfection of HUVECs with 4 μM anti-miR-31 inhibitor (n = 2) or anti-miR-Neg (n = 2) induced a ≥1.69-fold increase in PROX1 mRNA levels after 48 h. Data were normalized using β-actin and are shown as mean relative abundances ± the standard deviations. ***, P ≤ 0.001.

miR-31 overexpression in Xenopus embryos impairs lymphatic vessel sprouting from the lymph heart. Two-cell-stage Xenopus embryos were coinjected with pre-miR-31 precursor (50 ng) or pre-miR-Neg (100 ng) control molecules and 0.2 ng of β-Gal mRNA (lineage tracer) and raised to stage 39. (A and B) Lymphatic vascular system development was monitored by whole-mount ISH for prox1 (A) and vegfr3 (B). Pre-miR-31-injected embryos exhibited marked defects in lymphatic vascular development, the most striking of which was impaired lymphatic vessel sprouting (arrowheads) from the lymph heart (arrow). (C) pecam1 ISH showed that intersomitic vein growth (arrowheads) was unilaterally misguided or delayed in embryos with elevated levels of miR-31.