自噬调节电离辐射引起的神经胶质瘤细胞初级纤毛发生

Autophagy regulates ionizing radiation-induced primary ciliogenesis in glioblastoma cells

探讨自噬与电离辐射诱导人神经胶质瘤细胞初级纤毛发生的关系。神经胶质瘤细胞M059K和M059J进行10GyX射线照射或血清饥饿处理,免疫荧光法检测纤毛标志物Arl13b及基体结构蛋白γ-tubulin指示初级纤毛并统计纤毛发生率,免疫荧光法检测自噬标志物LC3联合蛋白质免疫印迹法检测p62蛋白表达量评估细胞自噬水平,利用氯喹(CQ)或雷帕霉素(RAPA)抑制或激活自噬水平并检测对细胞纤毛发生的影响。结果显示:M059K细胞中具有纤毛的细胞比例约为40%,X射线照射处理后3d上升至75%以上(p<0.01),但M059J细胞中纤毛细胞的比例(约7%)在X射线处理后无明显变化,然而血清饥饿处理3 d时M059K和M059J中纤毛细胞的比例分别上升至约80%(p<0.01)和50%(p<0.01)。血清饥饿导致两种细胞的自噬水平均显著升高,但X射线照射仅引起M059K细胞自噬水平升高而对M059J细胞影响不明显。使用RAPA激活细胞自噬能够显著提高两种细胞中纤毛细胞的比例,而使用CQ抑制自噬能够降低X射线照射引起的M059K细胞纤毛发生或血清饥饿引起的M059J细胞纤毛发生。这些结果表明M059K和M059J细胞在X射线处理后自噬激活水平的差异可能是导致两种细胞在照射后纤毛发生情况不同的重要原因,提示自噬在电离辐射诱导神经胶质瘤细胞纤毛发生中发挥重要调节作用。

The role of autophagy in ionizing radiation(IR)-induced primary ciliogenesis in glioblastoma cells was explored.M059K and M059J cells underwent treatment with either 10 Gy of X-rays or serum starvation(SS)for three days.Subsequently,the primary cilia were examined through immunofluorescence staining targeting ciliary markers Arl13b andγ-tubulin.Cellular autophagy levels were estimated by immunofluorescence staining for the autophagy marker LC3 and immunoblotting analysis of p62.To assess primary ciliogenesis,M059K and M059J cells were treated with chloroquine(CQ)or rapamycin(RAPA),which respectively suppressed or activated cellular autophagy.The findings revealed that in M059K,the proportion of ciliated cells was approximately 40%,and this proportion increased to over 75%on day 3 post-irradiation in cells treated with 10 Gy of X-rays(p<0.01).However,the frequency of primary cilia in M059J cells(~7%)showed no obvious changes before and after X-ray irradiation.Interestingly,the proportion of ciliated cells boosted to approximately 80%(p<0.01)and 50%(p<0.01)in M059K and M059J cells starved for 3 d,respectively.Serum starvation(SS)elevated cellular autophagy levels in both cell lines,whereas X-ray irradiation enhanced autophagy levels only in M059K but not in M059J cells.Moreover,activation of autophagy using RAPA facilitated ciliogenesis in both cell lines,while the suppression of autophagy with CQ mitigated IR-induced cilia formation in M059K and SS-induced cilia formation in M059J cells.In summary,these results demonstrate that different cellular autophagy responses to IR in M059K and M059J cells participated in primary ciliogenesis elicited by IR exposure.