Fig 2: TIM-1 expression in hBMECs was reduced by miR-142 and increased by miR-142 inhibitor. TIM-1 mRNA levels were measured using RT-qPCR in hBMECs transfected with miR-142 mimic, inhibitor, or scramble (negative control) for 48 h, normalizing to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All experiments were performed at least in triplicate; the box-and-whiskers graph shows the medians and the 5th–95th percentiles; * p < 0.01 vs. miR scramble. Sequences of oligonucleotide primers are reported in Table 1.

Fig 3: Identification of miR-142 as a specific modulator of TIM-1; the complementary nucleotides between the target region of TIM-1 3'-UTR and hsa-miR-142-3p are highly conserved across different species.

Fig 4: The targeting of TIM-1 by miR-142 significantly reduced endothelial permeability. Endothelial leakage triggered by 48 h incubation with TIM-1 agonist TIMD4 (10 or 100 ng/mL; BioLegend San Diego, CA, USA) was measured in hBMECs transfected with miR-142 mimic, miR scramble, or by combining miR-142 mimic and TIM-1 overexpression (pcDNA3.1-TIM-1 plasmid; GenScript, Piscataway, NJ, USA). All experiments were performed at least in triplicate; in the violin plot, median (solid line) and quartiles (dotted lines) are indicated; * p < 0.01 vs. miR scramble, # p < 0.01 vs. vehicle.

Fig 5: The results obtained via RT-qPCR were confirmed in terms of protein levels (Biorbyt; #orb382613; Cambridge, UK), as shown in the representative immunoblots in panel (A) and their quantification in panel (B). All experiments were performed at least in triplicate; the box-and-whiskers graph indicates the medians and the 5th–95th percentiles; *: p < 0.01 vs. miR scramble.