Knock-down of UHRF1 expression enhances the radiosensitivity of Eca-109 cells / 西安交通大学学报(医学版)

ABSTRACT

Objective To investigate the effect of ubiquitin-like with PHD and ring finger domains 1 (UHRF1) on the radiosensitivity of esophageal squamous carcinoma cells and explore its mechanism. Methods The shRNA sequence was designed with human UHRF1 as the target, and the UHRF1-shRNA was transfected into Eca-109 cells mediated by lentivirus. The expressions of UHRF1 mRNA and protein before and after transfection were determined by fluorescence quantitative PCR and Western blotting. The radiosensitivity of Eca-109 cells after down-regulation of UHRF1 gene was assessed by colony formation assay. The effect of silencing UHRF1 on the apoptosis of Eca-109 cells before and after X-ray irradiation was detected by flow cytometry. Western blotting was used to detect the effects of UHRF1 silencing and X-ray irradiation on the expressions of apoptosis-related proteins Bcl-2, caspase-3 and proteins related to AKT/mTOR signaling pathway in Eca-109 cells. Results We successfully constructed an Eca-109 shUHRF1 cell line with UHRF1 stablely down-regulated. Colony formation assays showed that the radiosensitivity of Eca-109 cells was increased after down-regulation of UHRF1 expression compared with the NC group. The proportion of apoptotic cells in Eca-109 cells was increased, while this change was more obvious after irradiation. Compared with NC group, down-regulation of UHRF1 expression increased the level of active caspase-3 protein and decreased the level of the apoptosis inhibitory protein Bcl-2; the expression levels of p-AKT and p-mTOR protein were decreased. The changes in these four proteins in Eca-109 cells with down-regulation of UHRF1 combined with 6 Gy X-ray irradiation were more obvious compared with the NC group. Conclusion UHRF1 knock-down increased the radiosensitivity of ESCC via inhibiting the Akt/mTOR signaling pathway activity and inducing the apoptosis of the cells.