PNKP沉默对骨肉瘤放疗的增敏作用

PNKP silencing increases radiosensitivity of osteosarcoma cells in vitro

目的研究多聚核苷酸激酶/磷酸酶(polynucleotide kinase/phosphatase,PNKP)在放疗诱导的细胞DNA损伤、细胞凋亡、细胞增殖和细胞周期中的调节作用。方法将U2OS细胞系分为3组:骨肉瘤细胞对照组、阴性si RNA转染组(si C)和PNKP si RNA转染组(si PNKP)。Western blot检测0、1、2、4、6、8 Gy 6个剂量水平γ-射线照射后U2OS细胞中PNKP的变化,CCK-8分析不同剂量γ-射线照射后细胞的存活率,彗星实验检测辐射后骨肉瘤细胞DNA的损伤,MTT法检测辐射后细胞增殖情况,流式细胞仪分析辐射后细胞凋亡、线粒体膜电位和细胞周期的变化。结果 4 Gy剂量的γ-射线可以显著降低骨肉瘤细胞U2OS中PNKP的表达(55.17%,P<0.01)和细胞的存活能力(与对照组相比下降50.16%,P<0.01)。与单独辐射组(si C+IR)对比,PNKP沉默可以抑制辐照后细胞的生长(抑制率增加到129.61%,P<0.01),增加DNA的损伤(DNA迁移量增加58.94%,P<0.01),使细胞周期停滞在S期,促进细胞凋亡以及降低线粒体膜电位水平。结论 PNKP沉默可以增加骨肉瘤细胞放射治疗的敏感性。

Objective To investigate the impact of polynucleotide kinase/phosphatase （PNKP） silencing on radiation-induced DNA damage, apoptosis, cell proliferation and cell cycle in osteosarcoma cells. Methods U2OS cells were divided into control （Ctrl） group, negative siRNA （siC） group and siPNKP group. After different doses （0, 1, 2, 4, 6 and 8 Gy） of γ-ray irradiation, the changes of PNKP level and cell survival rate were determined using Western blotting and CCK-8 assay. After irradiation at the best dose, DNA damage and cell proliferation were evaluated by Comet assay and MTT assay, respectively. Apoptosis, mitochondrial membrane potential and cell cycle were analyzed by flow cytometry. Results Irradiation of 4-Gy ~/-rays to osteosarcoma cells significantly reduced PNKP expression （ 55. 17% , P 〈 0.01 ） and cell viability （50. 16% , P 〈 0.01 ） compared with the Ctd group. PNKP silencing inhibited the cell growth （inhibitory rate reached to 129.61% , P 〈 0.01 ） and increased DNA damage （ DNA migration amount increased by 58.94% , P 〈 0.01 ） after irradiation. It also could arrest the cell cycle in S phase, accelerate apoptosis and reduce mitochondrial membrane potential as compared with the siCtrl ＋ IR group. Conclusion PNKP silencing increases osteosarcoma cell sensitivity to radiation therapy.