盐诱导激酶2对电离辐射诱导的自噬与凋亡的影响

Effects of salt-inducible kinase 2 on autophagy and apoptosis induced by ionizing radiation

为探索盐诱导激酶2(salt-inducible kinase 2,SIK2)对电离辐射引起的自噬与凋亡的影响,以期为肿瘤的放射治疗提供新的思路,以慢病毒颗粒LV-GFP和LV-SIK2体外转染He La细胞,48 h后用荧光显微镜观察转染是否成功,RT-PCR和Western blot检测转染细胞SIK2的干扰效果。通过Western blot检测SIK2低表达对8Gy60Coγ射线照射下细胞中自噬相关蛋白LC3-Ⅱ和P62表达的影响来观察自噬的变化;利用流式细胞术来检测SIK2低表达对8 Gy60Coγ射线照射下细胞凋亡的影响。结果表明,SIK2低表达的稳转细胞系被成功构建;8Gyγ射线照射后,SIK2低表达细胞系LC3-Ⅱ表达下调,P62表达上调,表明自噬被减弱;流式细胞术检测发现SIK2低表达细胞系在照后12 h FITC+PI-细胞比例就显著增加,表明SIK2低表达增加了细胞凋亡。因此,SIK2低表达会导致电离辐射引起的细胞自噬大大减弱,凋亡显著增加。

The purpose of this study is to explore the effects of salt-inducible kinase 2 （SIK2） on autophagy and apoptosis induced by ionizing radiation in the hope of providing a new idea for tumor radiotherapy. Lentivirus LV - GFP and LV - SIK2 were transfected into HeLa cells in vitro. The success of transfection was examined by fluorescence microscopy after 48 h. Real - time PCR and Western blot were used to measure the efficiency of the SIK2 knockdown. After exposure to 8 Gy ^60Co γ irradiation, the effects of the SIK2 knockdown on autophagy related protein LC3 - II and on the P62 expression were examined by Western blot to observe the changes of autophagy. Flow cytometry was performed to examine the effect of the SIK2 knockdown on the apoptosis after 8 Gy^60Co γ irradiation. The results showed that the SIK2 knockdown of stably transfected cell line was established successfully. In the down-regulating SIK2 cell line after 8 Gy γ irradiation, the expression of LC3 - II was down- regulated while the P62 expression was up-regulated, indicating that autophagy was reduced. Flow cytometric analysis showed that FITC ^＋/PI^- cells ratio was already increased significantly at 12 h post irradiation in the down-regulating SIK2 cell line, indicating that the SIK2 knockdown increased the cell apoptosis. In conclusion, the SIK2 knockdown could lead to a significant increase of cell apoptosis induced by ionizing radiation.