西罗莫司和饥饿处理对人A431细胞自噬水平的影响

目的 探讨经典自噬诱导剂西罗莫司和饥饿处理对人皮肤鳞状细胞癌细胞系A431自噬水平的影响。方法 分别将A431细胞和人宫颈癌细胞系HeLa分为对照组（单纯DMEM培养基处理）、二甲基亚砜（DMSO）组、20 nmol/L西罗莫司组、80 nmol/L西罗莫司组、平衡盐溶液（EBSS）组。作用4 h后，Western印迹检测A431细胞和HeLa细胞自噬微管相关蛋白轻链3A/3B（LC3A/B）、重组人γ?氨基丁酸受体相关蛋白（GABARAP）的表达水平。吖啶橙染色分析细胞自噬体表达水平。结果 Western印迹检测显示，对照组与DMSO组A431细胞中LC3A/B?Ⅱ与LC3 A/B?Ⅰ比值差异无统计学意义（P > 0.05）；20 nmol/L西罗莫司组、80 nmol/L西罗莫司组、EBSS组LC3 A/B?Ⅱ与LC3 A/B?Ⅰ比值较对照组均明显升高，差异均有统计学意义（P < 0.05）。HeLa细胞Western印迹检测结果与A431细胞类似。双变量相关性分析显示，HeLa细胞中LC3A/B?Ⅰ与GABARAP表达呈正相关（r = 0.869，95% CI：0.807 ~ 0.999，P = 0.051），LC3A/B?Ⅱ与GABARAP表达呈负相关（r = -0.742，95% CI：-0.982 ~ 0.406，P = 0.042）；A431细胞中LC3A/B?Ⅰ与GABARAP表达亦呈正相关（r = 0.837，95% CI：-0.173 ~ 0.989，P = 0.037），LC3A/B?Ⅱ与GABARAP表达呈负相关（r = -0.684，95% CI：-0.977 ~ 0.500，P = 0.047）。吖啶橙染色显示，A431细胞自噬阳性细胞比例在西罗莫司组（23.750% ± 0.260%）与EBSS组（32.450% ± 0.488%）同样高于对照组（15.987% ± 0.242%，均P < 0.05）；HeLa细胞自噬阳性细胞比例在西罗莫司组（33.307% ± 0.715%）和EBSS组（66.097% ± 1.141%）高于对照组（14.117% ± 0.295%，均P < 0.05）。结论 经典自噬诱导剂西罗莫司和饥饿能够上调A431细胞的自噬水平， GABARAP蛋白可能与LC3A/B高度相关。

Effects of sirolimus and starved culture on autophagy of human A431 cells

Zhang Yun, Yang Xiaowen, Xu Qianqian, Shi Xin, Chen Xu, Li Li, Xu Song, Huang Dan, Ju Mei, Chen Kun, Gu Heng Department of Dermatology, Second Affiliated Hospital of Soochow University, Suzhou 215004, China （Zhang Y ［current affiliation: Department of Dermatology, Yangzhou No.1 People′s Hospital, Yangzhou 225001, China］, Yang XW, Xu QQ, Shi X）; Department of Physical Therapy, Institute of Dermatology, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China （Chen X, Li L, Xu S, Huang D, Ju M, Chen K, Gu H） Corresponding authors: Chen Xu, Email: doctor_chx@126.com; Shi Xin, Email: shx9@163.com 【Abstract】 Objective To evaluate effects of sirolimus （a classic autophagy inducer） and starved culture on autophagy of a human cutaneous squamous cell carcinoma cell line A431. Methods Cultured A431 cells and HeLa （a human cervical carcinoma cell line） cells were both classified into 5 groups to be treated with DMEM alone （control group）, 0.1% dimethyl sulfoxide alone （DMSO group）, 20 nmol/L sirolimus （20-nmol/L sirolimus group）, 80 nmol/L sirolimus （80-nmol/L sirolimus group）, and Earle′s balanced salt solution （EBSS group） respectively. After 4-hour treatment, Western blot analysis was performed to measure the s of autophagy-related markers microtubule-associated protein 1 light chain 3A/3B （LC3A/B） and recombinant gamma-aminobutyric acid receptor associated protein （GABARAP）, and acridine orange staining to determine autophagy levels in these cells. Results As Western blot analysis showed, the ratio of LC3A/B -Ⅱ to LC3A/B -Ⅰ in A431 cells was similar between the control group and DMSO group （P > 0.05）, but significantly higher in the 20-nmol/L sirolimus group, 80-nmol/L sirolimus group and EBSS group than in the control group （all P < 0.05）. Western blot results from HeLa cells were similar to those from A431 cells. Bivariate correlation analysis revealed that the protein of GABARAP was positively correlated with that of LC3A/B -Ⅰ in both HeLa cells （r = 0.869, 95% CI: 0.807 - 0.999, P = 0.051） and A431 cells （r = 0.837, 95% CI: -0.173 - 0.989, P = 0.037）, but negatively correlated with that of LC3A/B-Ⅱ in both HeLa cells （r = -0.742, 95% CI: -0.982 - 0.406, P = 0.042） and A431 cells （r = - 0.684, 95% CI: -0.977 - 0.500, P = 0.047）. Acridine orange staining showed that the percentages of autophagosome-positive A431 cells and HeLa cells were significantly increased in both the 80-nmol/L sirolimus group （23.750% ± 0.260% and 33.307% ± 0.715% respectively） and EBSS group （32.450% ± 0.488% and 66.097% ± 1.141% respectively） compared with the control group （15.987% ± 0.242% and 14.117% ± 0.295%, respectively, all P < 0.05）. Conclusion The classic autophagy inducer sirolimus and starved culture can upregulate the autophagy level of A431 cells, and GABARAP may be highly correlated with LC3A/B.