氧化应激下黑素细胞自噬相关基因差异表达分析

摘要/Abstract

摘要： 目的探讨过氧化氢（H2O2）对黑素细胞自噬的影响及可能的调节机制。方法取对数生长期健康人黑素细胞，分为空白对照组（不予任何处理）、阳性对照组（100 nmol/L西罗莫司处理）和实验组（体积分数为10?7 ~ 10?3的H2O2处理），处理4 h后，使用CCK8法、流式细胞仪分别检测各组黑素细胞活性及凋亡率。吖啶橙染色检测自噬小泡，透射电镜下观察自噬小体，Western印迹检测自噬特异性蛋白Beclin 1、微管相关蛋白轻链3B（LC3B）。最后采用包含84个自噬相关基因的RT2 Profiler PCR Array筛选自噬相关的差异表达基因。结果黑素细胞分别经10?3、5 × 10?4、10?4、5 × 10?5、10?5、5 × 10?6、10?6 H2O2处理后，细胞增殖活性及凋亡率与空白对照组相比差异均有统计学意义（F值分别为286.95、301.23，均P＜0.05），且随H2O2体积分数升高，增殖活性降低，凋亡率升高。除5 × 10?6 H2O2组分别与10?5、10?6 H2O2组间相比细胞凋亡率差异无统计学意义外，上述各H2O2组间两两比较，黑素细胞增殖活性及凋亡率差异均有统计学意义（P < 0.05）。吖啶橙染色及电镜观察发现，10?5 H2O2、10?6 H2O2和西罗莫司处理的黑素细胞中有自噬小体形成。Western印迹显示，10?5 H2O2、10?6 H2O2和西罗莫司组黑素细胞Beclin 1表达量和LC3B?Ⅱ/LC3B?Ⅰ比率均较空白对照组显著升高（P＜0.05）。RT2 Profiler PCR Array结果显示，与空白对照组相比，10?5 H2O2组、10?6 H2O2组和西罗莫司组中ATG12、ATG3、ULK1、PIK3CG、PTEN、PIK3C3表达均显著上调，EIF2AK3表达显著下调；10?5 H2O2组和西罗莫司组mTOR表达显著下调，ULK2表达显著上调；10?6 H2O2组mTOR表达未发生明显改变, AMPK、JNK1表达显著上调。结论体积分数为10?5和10?6的H2O2均能有效诱导黑素细胞自噬，可能与影响相关信号分子表达相关。

Abstract: Gong Qingli, Li Xue, Ding Gaozhong, Ling Yuting, Zhao Wen′e, Xiong Xixi, Lu Yan Department of Dermatology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China （Gong QL, Li X, Ding GZ, Ling YT, Xiong XX, Lu Y）; Department of Analysis and Testing Center, Nanjing Medical University, Nanjing 210029, China （Zhao WE） Corresponding author: Lu Yan, Email: luyan6289@163.com 【Abstract】 Objective To evaluate the effect of hydrogen peroxide（H2O2） on autophagy in melanocytes, and to explore its possible regulatory mechanisms. Methods Normal human melanocytes at exponential growth phase were divided into several groups: blank control group receiving no treatment, positive control group treated with 100 nmol/L sirolimus solution, and experiment groups treated with H2O2 solution at different volume fractions of 10-7 - 10-3 respectively. After 4-hour treatment, cell counting kit-8 （CCK-8）assay and flow cytometry were performed to evaluate the cellular proliferative activity and detect apoptosis of melanocytes respectively. Acridine orange staining was performed to detect autophagosome formation, transmission electron microscopy to observe ultrastructural changes of autophagosomes, and Western blot analysis to measure the of autophagy-specific protein Beclin 1 and microtubule-associated protein 1 light chain 3B （LC3B）. A total of 84 autophagy-related genes were analyzed by RT2 Profiler PCR Array, so as to screen differentially expressed autophagy-related genes. Results After the treatment with H2O2 at different volume fractions of 10-3, 5 × 10-4, 10-4, 5 × 10-5, 10-5, 5 × 10-6 and 10-6, experiment groups showed significantly decreased cellular proliferative activity, but significantly increased apoptosis rate compared with the blank control group （F = 286.95, 301.23, respectively, both P < 0.05）. With the increase in volume fractions of H2O2, the cellular proliferative activity was significantly gradually decreased （P < 0.05）, while the apoptosis rate showed an opposite trend （P < 0.05）, except that the 5 × 10-6 H2O2 group showed no significant differences in the apoptosis rate compared with the 10-5 H2O2 group and 10-6 H2O2 group. Acridine orange staining and electron microscopy showed autophagosome formation in the 10-5 H2O2 group, 10-6 H2O2 group and positive control group. Western blot analysis revealed that Beclin1 and LC3B-Ⅱ/LC3B-Ⅰ ratio were significantly higher in the 10-5 H2O2 group, 10-6 H2O2 group and positive control group than in the blank control group （all P < 0.05）. RT2 Profiler PCR Array showed significant up-regulation of ATG12, ATG3, ULK1, PIK3CG, PTEN and PIK3C3 genes and significant down-regulation of EIF2AK3 gene in the 10-5 H2O2 group, 10-6 H2O2 group and positive control group compared with the blank control group. In the 10-5 H2O2 group and positive control group, the mTOR gene was significantly up-regulated, and the ULK2 gene was significantly down-regulated. The 10-6 H2O2 group showed no obvious changes in the of mTOR gene, but significant up-regulation of AMPK and JNK1 genes. Conclusion H2O2 at volume fractions of 10-5 and 10-6 can induce autophagy in melanocytes, likely by influencing the of some related signaling molecules.

龚晴丽李雪丁高中凌雨婷赵文娥熊喜喜鲁严. 氧化应激下黑素细胞自噬相关基因差异表达分析[J]. 中华皮肤科杂志, 2017, 50(8): 547-552.

参考文献 22

［1］	Namazi MR. Neurogenic dysregulation, oxidative stress, autoim⁃munity, and melanocytorrhagy in vitiligo: can they be interconnected?［J］. Pigment Cell Res, 2007, 20（5）: 360⁃363. DOI: 10.1111/j.1600⁃0749.2007.00408.x.
［2］	Prignano F, Pescitelli L, Becatti M, et al. Ultrastructural and functional alterations of mitochondria in perilesional vitiligo skin［J］. J Dermatol Sci, 2009, 54（3）: 157⁃167. DOI: 10.1016/j.jdermsci.2009.02.004.
［3］	李雪, 周梅华, 吴迪, 等. 白癜风皮损边缘黑素细胞线粒体超微结构的电镜观察［J］.中华皮肤科杂志, 2013, 46（9）: 636⁃639. DOI: 10.3760/cma.j.issn.0412⁃4030.2013.09.008.
［4］	Schallreuter KU, Gibbons NC, Zothner C, et al. Hydrogen peroxide⁃mediated oxidative stress disrupts calcium binding on calmodulin: more evidence for oxidative stress in vitiligo［J］. Biochem Biophys Res Commun, 2007, 360（1）: 70⁃75. DOI: 10.1016/j.bbrc.2007.05. 218.
［5］	Les JJ. Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging［J］. Rejuvenation Res, 2005, 8（1）: 3⁃5. DOI: 10.1089/rej.2005.8.3.
［6］	Swope VB, Medrano EE, Smalara D, et al. Long⁃term proliferation of human melanocytes is supported by the physiologic mitogens alpha⁃melanotropin, endothelin⁃1, and basic fibroblast growth factor［J］. Exp Cell Res, 1995, 217（2）: 453⁃459. DOI: 10.1006/excr.1995.1109.
［7］	Hu DN, McCormick SA, Seedor JA, et al. Isolation, purification and cultivation of conjunctival melanocytes［J］. Exp Eye Res, 2007, 84（4）: 655⁃662. DOI: 10.1016/j.exer.2006.12.003.
［8］	Li X, Wu D, Shen J, et al. Rapamycin induces autophagy in the melanoma cell line M14 via regulation of the levels of Bcl⁃2 and Bax［J］. Oncol Lett, 2013, 5（1）: 167⁃172. DOI: 10.3892/ol.2012.986.
［9］	Eisenberg⁃Lerner A, Bialik S, Simon HU, et al. Life and death partners: apoptosis, autophagy and the cross⁃talk between them［J］. Cell Death Differ, 2009, 16（7）: 966⁃975. DOI: 10.1038/cdd.2009.33.
［10］	Whittemore ER, Loo DT, Watt JA, et al. A detailed analysis of hydrogen peroxide⁃induced cell death in primary neuronal culture［J］. Neuroscience, 1995, 67（4）: 921⁃932.
［11］	Chen Y, Klionsky DJ. The regulation of autophagy ⁃ unanswered questions［J］. J Cell Sci, 2011, 124（Pt 2）: 161⁃170. DOI: 10.1242/jcs.064576.
［12］	Kabeya Y, Kamada Y, Baba M, et al. Atg17 functions in cooperation with Atg1 and Atg13 in yeast autophagy［J］. Mol Biol Cell, 2005, 16（5）: 2544⁃2553. DOI: 10.1091/mbc.E04⁃08⁃0669.
［13］	Jung CH, Jun CB, Ro SH, et al. ULK⁃Atg13⁃FIP200 complexes mediate mTOR signaling to the autophagy machinery［J］. Mol Biol Cell, 2009, 20（7）: 1992⁃2003. DOI: 10.1091/mbc.E08⁃12⁃1249.
［14］	Batty IH, Hickinson DM, Downes CP. Cross⁃talk between phospholipase C and phosphoinositide 3⁃kinase signalling pathways［J］. Biochem Soc Trans, 1997, 25（4）: 1132⁃1137. DOI: 10.1042/bst0251132.
［15］	Maehama T, Dixon JE. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylino⁃sitol 3,4,5⁃trisphosphate［J］. J Biol Chem, 1998, 273（22）: 13375⁃13378.
［16］	Ueno T, Sato W, Horie Y, et al. Loss of Pten, a tumor suppressor, causes the strong inhibition of autophagy without affecting LC3 lipidation［J］. Autophagy, 2008, 4（5）: 692⁃700.
［17］	Egan DF, Shackelford DB, Mihaylova MM, et al. Phosphorylation of ULK1 （hATG1） by AMP⁃activated protein kinase connects energy sensing to mitophagy［J］. Science, 2011, 331（6016）: 456⁃461. DOI: 10.1126/science.1196371.
［18］	Itakura E, Kishi C, Inoue K, et al. Beclin 1 forms two distinct phosphatidylinositol 3⁃kinase complexes with mammalian Atg14 and UVRAG［J］. Mol Biol Cell, 2008, 19（12）: 5360⁃5372. DOI: 10.1091/mbc.E08⁃01⁃0080.
［19］	Wei Y, Pattingre S, Sinha S, et al. JNK1⁃mediated phosphorylation of Bcl⁃2 regulates starvation⁃induced autophagy［J］. Mol Cell, 2008, 30（6）: 678⁃688. DOI: 10.1016/j.molcel.2008.06.001.
［20］	He C, Zhu H, Li H, et al. Dissociation of Bcl⁃2⁃Beclin1 complex by activated AMPK enhances cardiac autophagy and protects against cardiomyocyte apoptosis in diabetes［J］. Diabetes, 2013, 62（4）: 1270⁃1281. DOI: 10.2337/db12⁃0533.
［21］	Verfaillie T, Salazar M, Velasco G, et al. Linking ER stress to autophagy: potential implications for cancer therapy［J/OL］. Int J Cell Biol, 2010［2015⁃04⁃05］. http://www.ncbi.nlm.nih.gov,SSL+IJCB2010⁃930509.pdf. DOI: 10.1155/2010/930509.
［22］	Yorimitsu T, Klionsky DJ. Endoplasmic reticulum stress: a new pathway to induce autophagy［J］. Autophagy, 2007, 3（2）: 160⁃162.