Study On Effects And Mechanisms Of Long Intergenic Noncoding RNA-p21 Knockdown On Radiosensitivity Of Hypoxic Tumor Cells

Objective: This study aims to investigate the effect and mechanisms of lincRNA-p21 knockdown on radiosensitivity of hypoxic tumor cells through generating hypoxic tumor cells with stable integration of the lincRNA-p21 shRNA.Methods: The expression of lincRNA-p21 in human liver cancer cells SMMC7721 and glioma cells U251 MG cells were detected by qRT-PCR after 2, 4 and 6 Gy dose of X-ray irradiation and hypoxic(1% O2) treatment. Lentivirus vector carrying lincRNA-p21 shRNA or scrambled shRNA were constructed, then SMMC7721 and U251 MG cells with stable integration of the lincRNA-p21 shRNA or scrambled sequence were generated through lentiviral-mediated gene transfer. Hypoxic or normoxic tumor cell proliferation was evaluated by MTT. Cell cycle and apoptosis were analyzed by flow cytometry. Motility of cells were detected by wound healing assay. Radiosensitivity of tumor cells were measured by cloning formation assay. Monodansylcadaverine(MDC) staining and western blot were used to analyze autophagy.Results: LincRNA-p21 was induced in a dose-dependent manner in SMMC7721 and U251 MG cells treated by 2, 4 and 6 Gy dose of X-ray irradiation. After SMMC7721 and U251 MG cells were cultured under hypoxic(1% O2) conditions for 24 and 48 h, lincRNA-p21 was induced in a time-dependent manner. Stable integration of the lincRNA-p21 shRNA reduced the expression of lincRNA-p21 in hypoxic tumor cells. After SMMC7721 and U251 MG cells were cultured under hypoxic(1% O2) conditions for 48 h, lincRNA-p21 knockdown induced tumor cells G2/M phase arrest and apoptosis, inhibited cell proliferation and migration, reduced levels of GLUT1 and HIF-1α protein via ubiquitin proteasome pathway. However, lincRNA-p21 knockdown showed no significant effect on the proliferation,cell cycle, apoptosis and migration in human hepatoma cells and glioma cells under normoxic conditions. LincRNA-p21 knockdown reduced autophagy via HIF-1/Akt/mTOR/P70 pathway and enhanced radiosensitivity of hypoxic SMMC7721 and U251 MG cells. The radiosensitization ratio of hypoxic SMMC7721 and U251 MG cells were 1.23 and 1.31, respectively. Nevertheless, lincRNA-p21 knockdown do not change the radiosensitivity of tumor cells under normoxic conditions. Overexpression of HIF-1α increased autophagy of hypoxic tumor cells with stable integration of the lincRNA-p21 shRNA and reduced its radiosensitivity.Conclusions: Irradiation and hypoxia treatment elevated the expression of lincRNA-p21. Knockdown of lincRNA-p21 induced hypoxic tumor cells G2/M phase arrest, promoted apoptosis, decreased cell proliferation and motility and reduced autophagy through HIF-1/Akt/mTOR/P70 pathway. These results delineate a novel mechanism of lincRNA-p21 knockdown in enhancing hypoxic tumor cell radiosensitivity, which may provide a new radiation therapeutic target for human hepatocellular carcinoma and glioma.