SIRT6调控细胞糖代谢对非小细胞肺癌A549细胞放射敏感性的影响#br# #br#

目的 探讨沉默信息调节蛋白6（SIRT6）调控细胞糖代谢对非小细胞肺癌A549细胞放射敏感性的影响。方法 构建过表达SIRT6的腺病毒载体Ad SIRT6，采用荧光实时定量PCR（QPCR）检测经不同转染强度（0、25、50、100、200 pfu／细胞）Ad SIRT6转染24 h后的SIRT6 mRNA水平；根据实验设计分为对照组、空载体（Ad-null）组及过表达（Ad-SIRT6）组，Ad-null组和Ad SIRT6组的病毒浓度均为200 pfu／细胞；采用克隆形成实验检测3组经0、2、4、6、8和10 Gy X线照射后的存活分数（SF），根据多靶单击模型绘制细胞存活曲线并计算增敏比（SER）；流式细胞术检测3组经4 Gy X线照射48 h的细胞周期和凋亡情况；采用QPCR检测经4 Gy X线照射48 h各组丙酮酸激2（PKM2）、乳酸脱氢酶A（LDHA）和己糖激酶（HK）的表达水平；葡萄糖试剂盒检测转染12、24、36、48 h后的培养基葡萄糖消耗水平。结果QPCR检测显示，在25～200 pfu／细胞的范围内，随转染强度的增加，A549细胞的SIRT6 mRNA水平升高（P＜0001），0、25、50、100、200 pfu／细胞对应的SIRT6 mRNA水平依次为1.012±0.016、1.356±0.185、2.243±0.695、4.887±1.169和7.241±1.425，50、100、200 pfu／细胞对应的SIRT6 mRNA水平均高于对照组，差异有统计学意义（P＜0.05）。后续实验选择过表达效果最强的200 pfu／细胞转染量。克隆形成实验显示，Ad null组和对照组经0、2、4、6、8、10 Gy X线照射后SF的差异无统计学意义（P＞0.05）；而Ad SIRT6组经4～10 Gy X线照射后的SF均低于Ad null组和对照组（P＜005）。多靶单击模型拟合细胞存活曲线提示SER为1.76。与对照组和Ad null组比较，Ad SIRT6组的G0／G1期细胞比例和凋亡率升高，S期细胞比例及PKM2、LDHA和HK mRNA水平均降低，差异有统计学意义（P＜005）；Ad SIRT6组转染12、24、36、48 h的葡萄糖消耗量降低，低于对照组和Ad null组（P＜0.05）。结论 SIRT6过表达可抑制A549细胞糖酵解过程中关键酶生成来抑制糖酵解过程，同时具有放射增敏作用，并可导致G0／G1期阻滞和细胞凋亡。

Effects of SIRT6 on glucose metabolism and regulation of radiosensitivity in non small cell lung cancer A549 cells

ObjectiveTo investigate the effect of regulating glucose metabolism by silent information regulator 6 （SIRT6） on radiosensitivity of non small cell lung cancer A549 cells. MethodsThe adenovirus vector Ad SIRT6 expressing SIRT6 was constructed， and mRNA levels of SIRT6 were detected by fluorescence quantitative real time PCR （QPCR） at 24 h after transfection with different transfection intensities （0， 25， 50， 100， 200 pfu／cell） of Ad SIRT6. According to the experimental design， A549 cells were divided into control group， empty load（Ad null） group and over expression （Ad SIRT6） group， and the virus concentration of Ad null group and Ad SIRT6 group were 200 pfu／cell. The survival fraction （SF） of 3 groups after 0， 2， 4， 6， 8 and 10 Gy X ray irradiation were detected by colony formation assay， and the cell survival curve was drawn and the sensitivity enhancement ratio （SER） was calculated according to the multi target model. Flow cytometry was used to detect the cell cycle and apoptosis at 48 h in 3 groups after 4 Gy X ray irradiation. The mRNA levels of pyruvate dehydrogenase 2 （PKM2）， lactate dehydrogenase A （LDHA） and hexokinase （HK） in glycolysis were detected by QPCR after 48 h irradiation with 4 Gy X ray. Glucose levels were measured by glucose kit after transfection for 12， 24， 36 and 48 h. ResultsQPCR results showed that in the range of 25 200 pfu／cell， the mRNA levels of SIRT6 increased with the increasing intensity of transfection （P＜0001）. The mRNA levels of SIRT6 were 1012±0016， 1356±0185， 2243±0695， 4887±1169 and 7241±1425 for A549 cells transfected with 0， 25， 50， 100， 200 pfu／cell. The mRNA levels of SIRT6 were higher in 50， 100， 200 pfu／cell than in control group （P＜005）， and the follow up test selected the strongest expression of 200 pfu／cell. The clone formation experiment showed that there was no significant difference in SF between Ad null group and control group after 0， 2， 4， 6， 8 and 10 Gy X ray irradiation （P＞005）， while the SF in Ad SIRT6 group after 4 10 Gy X ray irradiation was lower than that in Ad null group and control group （P＜005）. The cell survival curve of the multi target model showed that the SER was 176. Compared with control group and Ad null group， the percentage of G0／G1 phase cells and apoptotic rate in Ad SIRT6 group were increased， while the percentage of S phase cells and the mRNA levels of PKM2， LDHA and HK were decreased （P＜005）. The glucose consumption of Ad SIRT6 group decreased after 12， 24， 36 and 48 h of transfection， lower than those of control group and Ad null group （P＜005）. ConclusionOverexpression of SIRT6 can inhibit the formation of key enzymes during glycolysis in A549 cells to inhibit glycolysis as well as radiosensitivity， and lead to G0／G1 arrest and apoptosis.