Objective: To investigate the effect of targeting the silent information regulator 2 hemolog 2 (SIRT2) expression on the apoptosis of drug-resistant AML cell line HL-60/A and its mechanism.
Methods: The expression of SIRT2 and antophagy-related protein LCT, P62 in HL-60/A and HL-60 was detected by Western blot, the effect of cytorabine on the apoptosis of HL-60/A cells was detected by using Annexin V/PI double staining after targeting inhibition of SIRT2 expression resulting from transfecting HL-60/A cells with SiRNA. The Western blot and transmission electray microscopy were used to detect the cell autophagy. To further clarify the role of autophragy in the regulatory effect of SIRT2 on the drug-resistance of HL-60/A cells, the autophagy-specific agonist, repamycin, was added into the cell culture medium after SIRT2-siRNA transfection. Then, the autophagy and apoptosis of HL-60/A were detected, respectively.
Results: The SIRT2 protein expression obviously increased in HL-60/A cells than that in HL-60 cells. Moreover, the expression rate of LC3II/I was higher, but P62 expression was lower in HL-60/A cells. After siRNA successfully transfecting into HL-60/A cells, quantitative PCR and Western blot should that the expression of SIRT2 significantly decreased. Meawhile, Western blot showed that the expression of LC3 II/I decreased, but P62 increased. Meanwhile, By TEM found that the number of autophagosome also decreased, suggesting the autophagy was inhibited after down-regulation of SIRT2. In addition, the drug senstivity of HL-60/A cells to cytarabine in siRNA-transfection group increased, and the apoptotic rate detected by Annexin V/PI double staining significantly increased. However, after co-culture with rapamycin, the suppressed autophagy in siRNA-trasfect HL-60/A cells was activated, leading to the reappearance of drug resistance of cells to cytarabine, and more significantly decrease of apoptotic rate.
Conclusion: The high expression of SIRT2 in HL-60/A cells activates the protective autophagy mechanism, which closely related with drug resistance.
题目: SIRT2通过自噬机制逆转HL-60/A耐药性的研究.
目的: 探讨靶向沉默信息调控因子2同源蛋白2（SIRT2）表达对急性髓系白血病（acute myeloid leukemia，AML）耐药细胞株HL-60/A凋亡的影响及其潜在机制.
方法: 采用Western.blot分别检测HL-60/A及对药物相对敏感的亲代细胞株HL-60中SIRT2及自噬相关蛋白LC3、P62的表达。采用siRNA转染HL-60/A细胞，靶向抑制SIRT2表达后，采用Annexin V/PI双染法检测阿糖胞苷对HL-60/A细胞细胞凋亡的影响；采用Western blot和透射电子显微镜检测HL-60/A细胞的自噬影响。为明确自噬在SIRT2对HL-60/A耐药调节中的确切作用，在SIRT2沉默后加入自噬特异性激动剂雷帕霉素，再分别检测对HL-60/A细胞自噬与凋亡的影响.
结果: HL-60/A 细胞中SIRT2、LC3 II/I蛋白表达量较HL-60细胞明显增高（P<0.05），但P62表达显著降低（P<0.05）。siRNA成功转染HL-60/A细胞后，SIRT2表达量显著降低（P<0.05）；LC3 II/I蛋白表达明显下降（P<0.05），同时P62蛋白表达上升；自噬体数目显著降低（P<0.05）。这提示，自噬受到抑制。SIRT2被沉默后， HL-60/A 对阿糖胞苷的药敏性增加，细胞凋亡率显著上升（P<0.05）。而当加入雷帕霉素后，siRNA转染组中受到抑制的细胞自噬被激活，HL-60/A细胞重新表现出对阿糖胞苷的耐药性，细胞凋亡率较其他组明显下降（P<0.05）.
结论: SIRT2的高表达激活了HL-60/A细胞保护性自噬机制，且与其耐药性密切相关.