PMC

A. Quantification by qRT-PCR of PAI-1 mRNA level after over-expression of either Pre-miR-421, Pre-miR-30c or a Pre-miR Negative control (Pre-Neg) in HUVEC cells. RPL32 mRNA level was used for normalization and data shown were expressed as percentage compared to Negative control (*, p<0.05 n = 4). B. Western-Blot and quantification of PAI-1 and GAPDH protein level after over-expression of either Pre-miR-421, Pre-miR-30c or both compared to Pre- Neg transfected cells. Data shown were normalized to GAPDH protein level and expressed as percentage compared to Negative control (n = 5 for miR-421 and miR-30c, n = 3 for miR-421+30c; *, p<0.05; **, p<0.01).

MiR-421 and miR-30c were detected by qRT-PCR in plasma samples from two groups of 20 patients either with low (1.6+/−1 ui/ml) or high (40.5+/−13 ui/ml) PAI-1 plasma levels. 40 fmol of synthetic cel-miR (39/54/238) were used for normalization. Median values are shown in black line (*: p<0.05).

A. Psicheck2 vector containing total 3′UTR SERPINE1 sequence fused to renilla luciferase was co-transfected with Pre-Neg, Pre-miR-30c, Pre-miR-421 or both Pre-miR-30c and Pre-miR-421. Graph shows renilla luciferase activity normalized to firefly luciferase activity and expressed as percentage of Pre-Neg transfected cells (n = 6, p<0.005,***). B. Psicheck2 vector containing total 3′UTR SERPINE1 sequence with the mutated rs1050955-A allele fused to renilla luciferase was co-transfected with Pre-Neg, Pre-miR-421, Pre-miR-30c or both. Graph shows renilla luciferase activity normalized to firefly luciferase activity and expressed as percentage of Pre-Neg transfected cells (n = 5 ** p<0.01). C. Comparison of miR-421 inhibitory effect on luciferase activity between SERPINE1 3′UTR wild-type or mutated at rs1050955 (n = 4).

The 3′UTR human SERPINE1 sequence is 1841 bp long. The rs1050955-A muted allele is shown in red bold. A. SERPINE1 DNA sequence showing the rs1050955 G/A polymorphism in position 1737. B. Representation of various miRNA seed region and their complementary sequence in SERPINE1 3′UTR 1728–1756 region. C. Alignment of miR-421 onto SERPINE1 3′UTR 1704–1760 region which includes two predicted binding sites, site 1 and site 2, complementary to the miR-421 seed sequence.

A. psicheck2 vector containing 3′UTR SERPINE1 sequence surrounding miR-30c predicted binding site or miR-421 predicted binding sites according to the allele present at rs1050955 fused to renilla luciferase were co-transfected with Pre-Neg, Pre-miR-30c or Pre-miR-421. Graph shows renilla luciferase activity normalized to firefly luciferase activity and expressed as percentage of Pre-Neg transfected cells (n = 4 for miR-30c and n = 5 for miR-421; *, p<0.05; **, p<0.01)). B. Various 3′UTR SERPINE1 sequence containing both miR-421 site 1 and site 2 predicted binding sites or mutation of each seed sequence binding sites were fused to renilla luciferase. Plasmids were transfected with Pre-Neg or Pre-miR-421. Graphs show renilla luciferase activity normalized to firefly luciferase activity and expressed as percentage of Pre-Neg transfected cells (n = 5 except for site1+2 1 mut 2 mut construct, n = 4; *, p<0.05; **, p<0.01).