Abstract:

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.