Effects of RNAi-mediated TUSC3 silencing on radiation-induced autophagy and radiation sensitivity of human lung adenocarcinoma cell line A549 under hypoxic condition

Abstract

This study examined the effects of RNAi-mediated TUSC3 silencing on radiation-induced autophagy and radiation sensitivity of human lung adenocarcinoma cell line A549 under hypoxic condition. Different CoCl₂ concentrations were used to treat A549 cells and establish a CoCl₂-induced hypoxic model of A549 cells. MTT and clone formation assays were used to determine the effects of different concentrations of CoCl₂ on the growth and proliferation of A549 cells treated by different doses of X-ray irradiation. The siRNA-expressing vector was transfected by liposomes and for silencing of TUSC3. Flow cytometry was used to measure cell cycle changes and apoptosis rate. Real-time quantitative polymerase chain reaction (qRT-PCR) assay was performed to detect the expression of TUSC3 mRNA. Western blotting was applied to detect the changes of TUSC3, LC3, and p62 proteins under different CoCl₂ concentrations and after siRNA silencing of TUSC3. The TUSC3 levels in A549 cells increased under hypoxic conditions in a dose-dependent manner (P < 0.05). Hypoxia inhibited the growth and proliferation of A549 cells and promoted apoptosis (P < 0.05). With an increasing dose of X-ray irradiation, A549 cells showed significantly increased growth and proliferation and decreased apoptosis (P < 0.05). After siRNA-TUSC3 was transfected by liposome, the TUSC3 level was substantially inhibited (P < 0.05). Silencing TUSC3 inhibited A549 cell growth and proliferation after radiotherapy under hypoxic condition, promoted apoptosis, increased G0/G1 phase cells, and reduced S phase cells (all P < 0.05). Hypoxia and radiation along with different CoCl₂ concentrations could induce cell autophagy, which increased with concentration and dose, while silencing the TUSC3 gene inhibited autophagy (all P < 0.05). RNAi silencing of TUSC3 inhibited growth and proliferation, while enhanced apoptosis and radiation sensitivity of hypoxic A549 lung adenocarcinoma cells.

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