角蛋白18磷酸化增加结肠癌HCT116细胞自噬并提高其对奥沙利铂的敏感性  

Keratin 18 phosphorylation increases autophagy of colorectal cancer HCT116 cells and enhanced its sensitivity to oxaliplatin

作　　者：闫晓东[1,2] 石英[3] 冦卜心 朱振宇[1] 柴杰[1] 陈德喜[3] 郭洪亮[1] 

机构地区：[1]山东省肿瘤医院外科四病区,山东济南250117 [2]济南大学山东省医学科学院医学与生命科学学院,山东济南250022 [3]首都医科大学附属北京佑安医院感染科,北京100069

出　　处：《细胞与分子免疫学杂志》2016年第1期34-38,43,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81100288);国家科技支撑计划(2012BAI15B08);山东省自然科学基金(ZR2013HL045)

摘　　要：目的研究奥沙利铂(OXA)作用下角蛋白18(K18)磷酸化与结肠癌HCT116细胞自噬和凋亡的关系并分析其可能机制。方法 HCT116细胞分别转染空载质粒、野生型K18和33、52磷酸化位点突变的K18质粒(Ser33/52A),用60μmol/L的OXA处理细胞,加入自噬抑制剂3-甲基腺嘌呤(3-MA)或细胞自噬诱导剂雷帕霉素处理细胞。异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)双标记结合流式细胞术以及钙黄绿素乙酰甲酯/碘化丙啶(calcein-AM/PI)染色法分析K18及其突变体对细胞凋亡的影响;Western blot法检测K18的磷酸化水平、自噬相关蛋白微管相关蛋白1轻链3(LC3)、beclin-1的表达。结果 Ser33/52A质粒的转染可以明显降低K18的磷酸化水平,经过OXA处理后,转染K18质粒组的HCT116细胞的凋亡率显著高于空载质粒转染对照组,而Ser33/52A质粒转染的HCT116细胞的凋亡率明显低于空载体和K18质粒转染的细胞凋亡率,K18质粒转染组细胞自噬明显高于空载转染对照组,而Ser33/52A质粒转染组自噬水平明显降低。结论 K18过表达促进HCT116细胞自噬及其对OXA的敏感性,而K18 Ser33和Ser52磷酸化水平的降低则抑制自噬并降低HCT116细胞的凋亡率。Objective To study the correlation between the phosphorylation of keratin 18（ K18） and the autophagy and apoptosis of HCT116 cells under the effect of oxaliplatin（ OXA） and investigate its possible mechanism. Methods HCT116 cells were transfected with empty plasmid,wild-type K18 expression plasmid and 33,52 phosphorylation site mutated K18（ Ser33 /52A） expression plasmid separately,and all cells were then treated with 60 μmol/L OXA,followed by supplementation of autophagy inhibitor 3-methyladenine（ 3-MA） or autophagy inducer rapamycin. FITC-conjugated annexin V and propidium iodide（ PI） double staining combined with flow cytometry,calcein-AM / PI staining were used to analyze the effects of K18 and its mutants on cell apoptosis; Western blotting was performed to detect the expressions of K18 phosphorylation,autophagy related proteins microtubule associated protein 1 light chain 3（ LC3） and beclin-1. Results Transfection of Ser33 /52 A plasmid significantly reduced the level of K18 phosphorylation. After treated with OXA,the apoptosis rate of K18 plasmid transfected group was significantly higher than that of empty plasmid transfected group,while the apoptosis rate of Ser33 /52 A plasmid transfected HCT116 cells was significantly lower than that of empty plasmid or K18 plasmid transfected group. Compared with empty plasmid group,the autophagy of K18 plasmid transfected group was significantly promoted,while the autophagy in Ser33 /52 A plasmid transfected group was significantly inhibited. Conclusion K18 overexpression enhanced the autophagy in HCT116 cells and increased its sensitivity to OXA. The decrease of K18 ser33 and ser52 phosphorylation inhibited autophagy and decreased apoptosis of HCT116 cells.

关 键 词：结肠肿瘤 奥沙利铂 HCT116细胞 角蛋白18 磷酸化 细胞凋亡 

分 类 号：Q279[生物学—细胞生物学] R735.3[医药卫生—肿瘤]