Objective

2

To explore the effect of Shidao Tongjie Formula on angiogenesis of HUVECs induced by exosomes derived from Eca‑109 esophageal cancer cells.

Methods

2

Twenty male SD rats were randomly divided into the Shidao Tongjie Formula drug serum group and the blank serum group， with 10 rats in each group. Two groups of rats were respectively administered 51.3 g/kg of the Shidao Tongjie Formula and the same volume of 0.9% sodium chloride solution by gavage to prepare the drug serum. Eca-109 cells were treated with 20% exosome-free blank serum or 20% Shidao Tongjie Formula drug serum （volume fraction）. The exosomes derived from Eca-109 cells （Eca-109 exosomes） as well as the exosomes derived from Eca-109 cells that had been treated with the Shidao Tongjie Formula drug serum （ Shidao Tongjie Formula exosomes） were isolated by ultracentrifugation method. The exosomes were identified by electron microscopy， nanoparticle tracking analysis （NTA） and Western blot. Real-time fluorescence quantitative reverse transcription polymerase chain reaction （RT-qPCR） was used to detect the expression of long non-coding RNA zinc finger structure antisense transcript 1 （LncRNA ZFAS1） in exosomes. Exosomes were co-cultured with HUVECs， and the uptake of exosomes by HUVECs was

observed using immunofluorescence staining. The effect on the angiogenesis of HUVECs was evaluated by 5-ethynyl- 2'-deoxyuridine （EdU） and tube formation assays. LncRNA ZFAS1 and

Vascular endothelial growth factor A （

VEGFA

） mRNA in HUVECs were measured by RT-qPCR， and VEGFA protein in HUVECs was determined by Western blot.

Results

2

The results of electron microscopy， NTA and Western blot for the marker proteins of differentiation cluster 63 （CD63）， tumor susceptibility gene 101 （TSG101）， and calnexin indicated that both groups of extracts were identified as exosomes. It was observed that both groups of exosomes were taken up by HUVECs under fluorescence microscope. The expression of LncRNA ZFAS1 in Eca-109 cell derived exosomes was significantly decreased by Shidao Tongjie Formula （

P<

0.01）. Compared with the control group， the proliferation and tube formation of HUVECs induced by Eca-109 cells derived exosomes were significantly promoted （

P<

0.05，

P<

0.01）， and the expressions of LncRNA ZFAS1，

VEGFA

mRNA and VEGFA protein in HUVECs cells were significantly upregulated （

P<

0.05）. While Shidao Tongjie Formula could significantly reduce the ability of Eca‑109 cells derived exosomes to induce the proliferation and tube formation of HUVECs （

P<

0.05，

P<

0.01）， and significantly downregulate the expressions of LncRNA ZFAS1，

VEGFA

mRNA and VEGFA protein in HUVECs cells （

P<

0.05）.

Conclusions

2

Eca-109 cells derived exosomal LncRNA ZFAS1 can promote angiogenesis in HUVECs. Shidao Tongjie Formula may inhibit esophageal cancer angiogenesis by reducing Eca-109 cells derived exosomal LncRNA ZFAS1 expression， downregulating LncRNA ZFAS1 and VEGFA expression in HUVECs， and suppressing HUVECs proliferation and tube formation.