miR-193a-3p interaction with HMGB1 downregulates human endothelial cell proliferation and migration

Circulating endothelial colony forming cells (ECFCs) contribute to vascular repair where they are a target for therapy. Since ECFC proliferative potential is increased in cord versus peripheral blood and to define regulatory factors controlling this proliferation, we compared the miRNA profiles of cord blood and peripheral blood ECFC-derived cells. Of the top 25 differentially regulated miRNAs selected, 22 were more highly expressed in peripheral blood ECFC-derived cells. After validating candidate miRNAs by q-RT-PCR, we selected miR-193a-3p for further investigation. The miR-193a-3p mimic reduced cord blood ECFC-derived cell proliferation, migration and vascular tubule formation, while the miR-193a-3p inhibitor significantly enhanced these parameters in peripheral blood ECFC-derived cells. Using in silico miRNA target database analyses combined with proteome arrays and luciferase reporter assays of miR-193a-3p mimic treated cord blood ECFC-derived cells, we identified 2 novel miR-193a-3p targets, the high mobility group box-1 (HMGB1) and the hypoxia upregulated-1 (HYOU1) gene products. HMGB1 silencing in cord blood ECFC-derived cells confirmed its role in regulating vascular function. Thus, we show, for the first time, that miR-193a-3p negatively regulates human ECFC vasculo/angiogenesis and propose that antagonising miR-193a-3p in less proliferative and less angiogenic ECFC-derived cells will enhance their vasculo/angiogenic function.

Cells were analyzed on a BD LSR II flow cytometer using FACS Diva software (BD Biosciences) as previously described 1 . Cells were stained using single conjugated antibodies and then incubated with 1:1000 DAPI (Molecular Probes, Paisley, Scotland) to select viable cells prior to CD antigen analysis.

Western blot
Western blot was performed with protein extracts from CB and PB ECFC-derived cells transfected with miR-193a-3p mimic and mimic non-targeting control. Cell pellets were lysed on ice for 30 min in lysis buffer containing 20 mM Tris-Cl, 137 mM NaCl, and 10% glycerol with x1 protease inhibitors. The supernatant collected after centrifugation was quantitated, and 50 µg of protein extract was loaded onto a NuPage 4-12% Tris Bis gel (Invitrogen). The following primary mouse antibodies were used: HYOU1 (clone 6F7; Abnova Corporation, Taipei City, Taiwan) and HMGB1 (clone EPR3507; Abcam, Cambridge, UK) for 1 h at room temperature.
Respective secondary antibodies were used and detection of positive signal was achieved using Odyssey CLx for Infrared Fluorescent imager plus (Licor Biosciences). Normalization was performed using antibody against alpha tubulin at 1:5000 (BD Biosciences).

Reverse transcription PCR and real time-quantitative-PCR (miRNA)
Isolated RNAs were converted to cDNA using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and 20ng RNA. Mature human microRNA expression of miR-193a-3p, miR-34a, miR-376a, miR-21, let-7c and miR-1908 was determined by real-time qPCR using TaqMan microRNA assays that contains target-specific stem-loop reverse transcription primers (Applied Biosystems; Assay 002250 for mir-193a-3p; Assay 002316 for miR-34a; Assay 000565 for miR-376a ;Assay 000397 for miR-21; Assay 000379 for let-7c and Assay 121109_mat for miR-1908) and performed on ViiA 7 q-RT-PCR (Life Technologies, Carlsbad, CA, USA). Fold changes in microRNA expression were determined by the 2 -ΔΔCt method, normalizing the results to expression of the control RNU 44 2 . Each PCR reaction was carried out in triplicate according to Taqman MicroRNA assay instructions and on a 96-well optical plate performed at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 60 seconds.

Reverse transcription PCR and real time-quantitative-PCR (mRNA)
Isolated RNAs were converted to cDNA using TaqMan using the High-Capacity RNAto cDNA Kit (Applied Biosystems) using 40ng RNA. mRNA expression of HMGB1 and HYOU1 was determined by real-time q-RT-PCR using TaqMan Gene Expression Assay primers (Life Technologies; HMGB1: Hs01590761_g1, HYOU1: Hs00197328_m1) and performed on ViiA 7 q-RT-PCR (Life Technologies). Fold changes in mRNA expression were determined by the 2-ΔΔCt method, normalizing the results to expression of the control beta-2-microglobulin. Each PCR reaction was carried out in triplicate according to Taqman Gene Expression assay instructions and on a 96-well optical plate performed at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 60 seconds.

Proteome array analysis by mass spectrometry
CB ECFC-derived cells (n=3) were transfected in parallel with mimic non-targeting control (10nM) and miR-193a-3p mimic (10nM) for 48 hr. Cells were washed with cold PBS and centrifuges. Cell pellets were frozen at -80°C before samples were Twelve proteins were predicted to target miR-193a-3p using 5 target prediction sites:-Targetscan, miRanda, miRDB, miRWalk and RNAhybrid