p53及其凋亡刺激蛋白对结肠癌细胞自噬和凋亡影响机制探讨

Research of mechanism of p53and apoptosis stimulating protein on autophagy and apoptosis in colon cancer cell

目的探讨p53、p53凋亡刺激蛋白2(apoptosis stimulating protein 2of p53,ASPP2)及异构体ΔASPP2在p53缺陷结肠癌细胞系HCT116-/-细胞中对自噬及凋亡的影响。方法实验分为对照组、MMS组、MMS+p53组、MMS+ASPP2+p53组、MMS+ΔASPP2+p53组和MMS+ASPP2+ΔASPP2+p53组。应用ASPP2、ΔASPP2及p53质粒转染HCT116-/-细胞,同时转染绿色荧光GFP-LC3质粒于各组细胞中,经甲基磺酸(MMS)处理后用荧光显微镜观察细胞自噬水平,蛋白质印迹法检测各组细胞自噬及凋亡相关蛋白表达水平,Calcein AM/PI染色检测细胞凋亡水平。结果自噬水平对照组为(0.63±0.14)%,MMS组为(10.48±0.58)%,MMS+p53组为(5.45±0.41)%,MMS+ASPP2+p53组为(3.43±0.35)%,MMS+ΔASPP2+p53组为(14.84±0.64)%,MMS+ASPP2+ΔASPP2+p53组为(12.29±0.58)%,差异有统计学意义,F=1 182.364,P<0.001;与MMS组比较,MMS+p53组和MMS+ASPP2+p53组自噬水平下降,P值均<0.001;与MMS+p53组相比,MMS+ASPP2+p53组自噬水平降低,P<0.001;MMS+ΔASPP2+p53组自噬水平较MMS+p53处理组增加,P<0.001;MMS+ASPP2+ΔASPP2+p53组自噬水平较MMS+ASPP2+p53组增加,P<0.001。凋亡率比较,对照组为(2.09±0.54)%,MMS组为(16.79±0.69)%,MMS+p53组为(33.70±1.16)%,MMS+ASPP2+p53组为(50.61±1.17)%,MMS+ΔASPP2+p53组为(21.32±1.18)%,MMS+ASPP2+ΔASPP2+p53组为(35.22±1.06)%,差异有统计学意义,F=2 571.942,P<0.001;MMS+p53和MMS+ASPP2+p53组凋亡较MMS组增加,P值均<0.001;与MMS+p53组相比,MMS+ASPP2+p53组凋亡增加,P<0.001;MMS+ΔASPP2+p53组凋亡较MMS+p53组减少,P<0.001;MMS+ASPP2+ΔASPP2+p53组凋亡较MMS+ASPP2+p53组也有所降低,P<0.001。结论 ASPP2能够增强p53的自噬抑制作用,并增强p53促细胞凋亡作用,ΔASPP2能够抑制ASPP2与p53的功能,具有促进细胞自噬,抑制细胞凋亡的作用。

OBJECTIVE To investigate the role of p53,SPP2 and isoform AASPP2 on cell autophagy and apoptosis in non-p53-expressed colon cancer cell line HCTll6-/- cells. METHODS The experiments were divided into 6 groups, including MMS group,MMS＋p53 group,MMS＋ASPP2＋p53 group,MMS＋ASPP2＋p53 group,MMS＋ASPP2＋AS- PP2 ＋ p53 group. HCT116 -/ cells were transfected with ASPP2, AASPP2, p53 and GFP-LC3 plasmids, then treated with methanesulfonie acid （MMS）. The cell autophagy was observed with immunofluorescence. The expression of autophagy and apoptosis-related protein were detected by using Western blotting. Apoptosis was observed by using Calcein AM/PI. RESULTS The rate of autophagy was （0. 63±0. 14）G in the control group, （10. 48±0. 58）% in the MMS group, （5. 454±0.41）G in the MMS＋p53 group,（3.43±0.35）% in the MMS＋ASPP2＋p53 group, （14.84±0. 64）G in the MMS＋AASPP2 ＋p53 group, and （ 12.29 ± 0. 58）% in the MMS 4- ASPP2 ＋ AASPP2 ＋ p53 group. The difference was statistically significant （F= 1 182. 364, P〈0. 001）. Compared with the MMS group, autophagy was decreased in the MMS ＋p53 group and MMS＋ ASPP2＋ p53 group （P〈0. 001）. Compared with the MMS＋ p53 group, autophagy was de- creased in the MMSq-ASPP24-p53 group （P〈0. 001）,and autophagy was increased in the AASPP2＋p53 group （P〈 0. 001）. Compared with the MMS＋p53＋ASPP2 group,autophagy was increased in the MMS＋p53＋ASPP2＋ AASPP2 group. The rate of apoptosis was （2.09 ± 0.54）% in the control group, （16.79±0.69） % in the MMS group, （33.70 4± 1.16）% in the MMS＋p53 group,（50.61±1.17）% in the MMS＋ASPP2＋p53 group,（21. 324±1.18）% in the MMS＋AASPP＋p53 group,and （35.22±1.06）% in the MMS＋ASPP＋△ASPP2＋p53 group. The difference was statistically significant （F=2 571. 942 ,P〈0. 001）. Compared with the MMS group,apoptosis was increased in the MMS--p53 group and MMS＋ASPP2--p53 group （P〈0. 001）. Compared with the MMS＋p53 group,apoptosis was increased in the MMS 4-ASPP2＋p53 group （P〈0. 001） and apoptosis was decreased in the group of AASPP＋p53 （P〈0. 001）. Compared with the MMS＋p53＋ASPP2 group,apoptosis was decreased in the group of MMSq-p53＋ASPP24-AASPP2. CONCLU- SIONS ASPP2 can enhance the function of p53 that inhibiting cell autophagy and promoting apoptosis,but AASPP2 can inhibit the function of ASPP2 and p53. It has the function of promoting cell autophagy and suppressing cell apoptosis.