腺病毒介导的白细胞介素37对非小细胞肺癌A549细胞生长及放射敏感性的影响

Effects of adenovirus-mediated interleukin-37 on the growth and radiosensitivity of non-small cell lung cancer cell line A549

目的研究腺病毒介导的白细胞介素37(IL-37)对人非小细胞肺癌(NSCLC)A549细胞生长及放射敏感性的影响,探讨IL-37作为新型放疗增敏剂的可能性。方法选择人NSCLC细胞株A549,将A549细胞分为3组:正常A549细胞(对照组)、空病毒转染的A549细胞[NC组,细胞感染复数(MOI)为100]、Ad-IL-37组(含IL-37的腺病毒转染A549细胞,MOI为100)。用蛋白质印迹法检测3组细胞IL-37蛋白表达情况;将3组细胞同时应用4 Gy照射剂量进行照射,四甲基偶氮唑盐(MTT)法检测吸光度(A)值,观察A549细胞增殖情况;流式细胞术观察受照射后各组A549细胞周期变化;蛋白质印迹法检测照射后各组细胞凋亡相关蛋白(bax、bcl-2、Caspase-3、Survivin)表达情况。结果对照组IL-37的相对表达水平为0.17±0.04,NC组为0.29±0.14,Ad-IL-37组为1.17±0.23(F=24.263,P=0.001)。照射后24 h时3组A值差异无统计学意义(F=2.587,P=0.160),照射后48、72、96、108 h时A值差异均具有统计学意义(F值分别为21.662、33.635、33.663、31.909,P值分别为0.005、0.001、0.001、0.001);照射后24 h时NC组和Ad-IL-37组细胞增殖抑制率差异无统计学意义(t=1.620,P=0.247),照射后48、72、96、108 h时两组细胞增殖抑制率差异均有统计学意义(t值分别为5.414、7.233、15.306、19.035,P值分别为0.032、0.019、0.004、0.003)。IL-37联合可影响细胞周期的变化,对照组S期细胞比例为(36.4±1.0)%,NC组为(31.3±0.6)%,Ad-IL-37组为(27.2±2.9)%(F=12.96,P=0.007),对照组G2/M细胞比例为(20.5±0.8)%,NC组为(24.7±2.9)%,Ad-IL-37组为(41.4±4.1)%(F=27.92,P=0.001)。IL-37能上调照射的A549细胞促凋亡相关因子bax、Caspase-3蛋白水平(F值分别10.31、14.51,P值均为0.01),下调抑凋亡因子bcl-2、Survivin蛋白的表达(F值分别8.95、5.52,P值分别0.02、0.04)。结论IL-37可抑制A549细胞生长,具有潜在的放射增敏作用,这一作用可能通过影响肿瘤细胞的凋亡产生。

Objective To analyze the effects of adenovirus-mediated interleukin-37(IL-37)on the growth and radiosensitivity of human non-small cell lung cancer(NSCLC)cell line A549,and to explore the possibility of IL-37 as a new radiosensitizer.Methods Human NSCLC cell line A549 was used as the research object.A549 cells were divided into three groups:normal A549 cells(control group),empty virus transfected A549 group[NC group,the multiple of infection(MOI)was 100],Ad-IL-37 group(A549 cells were transfected by adenovirus with IL-37,MOI was 100).The expression of IL-37 protein in three groups was detected by Western blot.The three groups were irradiated with 4 Gy irradiation dose at the same time.The methyl thiazolyl tetrazolium(MTT)method was used to detect the absorbance(A)values and observe the proliferation of A549 cells;flow cytometry was used to observe the phase changes of A549 cells in each group after irradiation;Western blot was used to detect the expressions of apoptosis-related proteins(bax,bcl-2,Caspase-3,and Survivin)in each group after irradiation.Results The expression level of IL-37 in the control group was 0.17±0.04,NC group was 0.29±0.14,and Ad-IL-37 group was 1.17±0.23(F=24.263,P=0.001);there was no statistical difference in A values among the three groups at 24 h after irradiation(F=2.587,P=0.160),while the differences of A values among the three groups at 48,72,96,108 h after irradiation were statistically significant(F values were 21.662,33.635,33.663,and 31.909,P values were 0.005,0.001,0.001,and 0.001,respectively).There was no significant difference in the cell proliferation inhibition rate between NC group and Ad-IL-37 group at 24 h after irradiation(t=1.620,P=0.247),while the differences were statistically significant at 48,72,96,108 h after irradiation(t values were 5.414,7.233,15.306,and 19.035,P values were 0.032,0.019,0.004,and 0.003,respectively).The combination of IL-37 and radiation could affect the cell cycle,the proportion of S-phase cells was(36.4±1.0)%in the control group,(31.3±0.6)%in the NC group,and(27.2±2.9)%in the Ad-IL-37 group(F=12.96,P=0.007),and the proportion of in G2/M-phase cells was(20.5±0.8)%in the control group,(24.7±2.9)%in the NC group,and(41.4±4.1)%in the Ad-IL-37 group,(F=27.92,P=0.001).IL-37 could up-regulate the expressions of pro-apoptotic factors bax and Caspase-3 proteins in A549 cells after irradiation(F values were 10.31 and 14.51,P values were both 0.01),and down-regulate the expressions of apoptotic factors bcl-2 and Survivin proteins(F values were 8.95 and 5.52,P values were 0.02 and 0.04).Conclusion IL-37 could inhibit the growth of human NSCLC cell line A549 and has potential radiosensitization effects,which may be caused by affecting the apoptosis of tumor cells.