关键词: 乏氧, 自噬, 放射敏感性, HIF-1&alpha, RNAi

Abstract:

Background and purpose: Hypoxia induced the decreased radiosensitivity of tumor cells, which was the cause of tumor radioresistance and relapse and metastasis. During the course, HIF-1a played the most important role in the regulation of hypoxia. However, it’s still unknown about the effect of HIF-1a on the radiosensitivity of hypoxia tumor cells and the relationship with autophagy. This study was to inhibit HIF-1α expression in hypoxic lung adenocarcinoma cell line A549 with RNA interference (RNAi), and explore its effect on hypoxic cell radiosensitivity and autophagy. Methods: Plasmids pHIF-1α-shRNA and Neg-shRNA (negative control) were constructed and transfected into hypoxic A549 cells, this positive clone was named A549/HIF-1α-shRNA. Clone formation array was applied to calculate the value of D0, SF2, SER. The expression of HIF-1α, LC3, c-parp was detected by Western blot. Results: The SF2 of hypoxic A549 cell was 0.62, which was higher than that of normoxic A549 cell, SER was 1.45. The level of LC3Ⅱ increased significantly and the level of c-parp decreased after the radiation of hypoxic A549 cell. The level of HIF-1a increased in hypoxic A549 cells. The expression of HIF-1α in hypoxic A549 cells was suppressed markedly after transfection of HIF-1α-shRNA; this clone was named A549/HIF-1α-shRNA. The SF2 and SER were significantly lower in A549/HIF-1α-shRNA cells, 0.45 and 0.72 respectively. Under the hypoxic condition and after the inhibition of HIF-1α, the expression of LC3Ⅱ decreased significantly and the expression of c-parp increased. Conclusion: We successfully established a cell model that HIF-1α expression was suppressed almost completely by RNAi. The inhibition of HIF-1α by shRNA may raise the radiosensitivity and decrease the autophagy of hypoxic A549 cells in vitro.

Key words: Hypoxia, Autophagy, Radiosensitivity, HIF-1α, RNAi