miR-320 regulates tumor angiogenesis driven by vascular endothelial cells in oral cancer by silencing neuropilin 1

Yi-Ying Wu1,5, Yuh-Ling Chen2,3,5, I-Shan Hsieh2, Yun-Chia Jao3, Kung-Chao Chang4, Tse-Ming Hong1*

*To whom correspondence should be addressed to Dr. Hong TM. Graduate Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan.+886 62353535 ext. 4259; Fax: +886 62359885; Email:

Supplementary methods

Cell adhesion assay

Cells (2.5x104cells per well) were incubated for 20 min at 37°C on 24 well tissue culture plates coated with 10 μg/mL fibronectin. Unbound cells were removed by washing with PBS twice. The bound cells were measured by 3-(4, 5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega, Madison, WI). Mixture with 15 μl MTS reagent and 135 μl phenol red free DMEM was added to each well and reacted for 90 minutes. The absorbance was measured at 490 nm in a plate reader. Each sample was assayed in triplicate.

Cell proliferation assay

After HUVECs were transfected with 20 nM of negative control or miR-320 precursor for 24 hours, 4x 104cells were seeded in 24-well plates and incubated for 72 hours. Cell number was measured by MTS assay every day as described above. Each sample was assayed in triplicate.

Supplementary figure legends

Supplementary Fig. 1: Schematic diagram of the 3rd predicted miR-320 binding sites in the NRP1 3’UTR region.Sequences were compared between maturemiR-320 and the wild-type or mutant (mt) putative target sites in the 3’UTR of NRP1 mRNA.

Supplementary Fig. 2: Representative expression levels of miR-320 (determined by in situ hybridization) and CD31 (determined by immunohistochemistry) in normal and tumor tissues of clinical specimens from another OSCC patient.

Supplementary Fig. 3: Representative expression levels of miR-320 (determined by in situ hybridization), CD31 and NRP1 (determined by immunohistochemistry) in normal and tumor tissues of clinical specimens from OSCC patients. Arrows indicate vessels. Scale bars: 100 μm.

Supplementary Fig.4:Representative expression levels of NRP1 (determined by immunohistochemistry) in normal and tumor tissues of clinical specimens from OSCC patients.

Supplementary Fig. 5:Effect of miR-320 on cell property of HUVECs. a Effect of miR-320 on the adhesive ability of HUVECs. Post 24 hour-transfection with 80 nM of control or antagomiR-320 (left) or 20 nM of control or miR-320 precursor (right), cells (2.5x104) were incubated for 20 min on 10 μg/ml fibronectin-coated substrate. Unbound cells were removed and amount of the bound cells were measured by MTS assay. The absorbance was measured at 490 nm in a plate reader. Adhesive abilities were normalized to the controls. The data are shown as the mean±SD of 3 wells. **P< 0.01; ***P< 0.001 by Student’s t-test. bEffect of miR-320 on cell proliferation of HUVECs. HUVECs transfected with 20 nM of negative control or miR-320 precursor (4x 104) were seeded in 24-well plates and incubated for 72 hours. Cell number was measured by MTS assay every day. Each data point represents the mean±SD of 3 wells.

Supplementary Fig. 6:miR-320 modulates angiogenesis in vitro. aLeft:HUVECs were transfected with 20 nM control or miR-320 precursor for 24 hours, after which capillary tube formation assays were performed. HUVECs transfected with the control or miR-320 were seeded onto Matrigel. Shown is a representative image at 12 hours after plating. Right:The relative tube length compared with that in the control group is presented as the mean± SD. *P < 0.05 by Student’s t-test. b Capillary tube formation assays of HUVECs transfected with 80 nM control antagomiR or antagomiR-320 (anti-320) were performed as described in a. Relative tube length in the anti-320 group compared with that in the control group is shown. ****P < 0.0001 by Student’s t-test

Supplementary Fig. 7: 50% to 60% confluence of HUVECs in EGM (growth) or EBM (basal) was exposed to 21% (normal) or 1% O2(hypoxia) for 48 hours. The miR-320 expression was assessed by quantitative real-time PCR. The miR-320 expression was normalized to that in normal-growth. The data are shown as the mean ± SD of 3 wells. *****P< 0.00001 by Student’s t-test.

Supplementary Fig. 8:Other candidate targets of miR-320 in HUVECs.aHUVECs were transfected with 20 nM miR-320 precursor or control (C) and 80 nM scrambled (control, C) or miR-320-specific antagomiR (antagomiR-320, anti-320). Twenty-four hours post transfection, the RNA levelsof NRP1, ITGB3, TGFBR1, EREG, IGF1 and β-actin were detected by RT-PCR. b The protein levelsof NRP1, integrin β3 and β-actin in cells, performed as a, were detected by western blot. β-actin was used as the internal control. The expression level wasnormalized to the internal control and relative expression level compared to the control group was shown.

1