KAI1对人胰腺癌MiaPaCa-2细胞自噬的影响  

Effect of KAI1 on autophagy of human pancreatic cancer cell line MiaPaCa-2

作　　者：吴春燕[1] 郭晓钟[1] 王华 

机构地区：[1]沈阳军区总医院消化内科,沈阳110015 [2]北京军事医学科学放射与辐射医学研究所

出　　处：《中华胰腺病杂志》2011年第6期400-403,共4页Chinese Journal of Pancreatology

基　　金：基金项目：国家自然科学基金（81071982、30470798）

摘　　要：目的观察感染Ad5-KAI1前后人胰腺癌细胞MiaPaCa-2自噬水平的变化，并初步探讨其机制。方法应用无KAI1表达的人胰腺癌细胞MiaPaCa2，通过感染带有KAI1目的基因的复制缺陷型腺病毒Ad5-KAll使细胞表达KAll，以Ad5-null感染作为阴性对照，亲本细胞为空白对照。用透射电镜观察细胞自噬小体，共聚焦显微镜观察自噬标志LC3颗粒。应用阻断剂PD98059和LY294002干预细胞，蛋白质印迹法检测自噬相关蛋白beclin1、LC3—Ⅱ、LC3-I及ERK-1／2、磷酸化ERK-1／2（P—ERK-1／2）、AKT、P—AKT的表达。结果以100MOIAd5-KAI1感染细胞24h，表达KAI1蛋白的细胞达（84．97±8．56）％；LC3颗粒从4个左右增加到20个以上；细胞线粒体肿胀、变性，胞质内双层膜样结构增加；Beclinl表达增加（1．4±0．3）倍，LC3-Ⅱ／LC3-I表达增加（8．00±2．78）倍。P13K阻断剂LY294002预处理细胞后可以有效地抑制MiaPaCa-2细胞AKT的磷酸化（2．756降至1．516），但不能抑制LC3—Ⅱ／LC3-I比值的增加（0．770增加到1．403）。ERK阻断剂PD98059预处理细胞后不仅可以有效地抑制MiaPaCa-2细胞ERK的磷酸化（1．637降至0．403），而且可以抑制beclin1蛋白表达的上调（2．377降至1．150）和LC3．11／LC3-I比值的增加（2．225降至0．680）。结论KAIl明显促进MiaPaCa2细胞内自噬，它是通过ERK而不是AKT磷酸化途径促进自噬的。Objective To study the change of autophagy of human pancreatic cancer cell MiaPaCa-2 before and after Ad5-KAI1 tranfeetiou, and to investigate the possible mechanism. Methods The MiaPaCa-2 ceils without KAI1 expression were infected with AdS-KAI1 with KAI1 target gene, and AdS-null was used as negative control, and parental cell was used as blank control. The formation of autophagosomes was observed by electromicroscopy. The green fluorescent protein-labeled light chain 3 （LC3） associations with autophagosome membranes was detected by confoeal microscopy. PD98059, LY294002 were applied to pre-treat the cells. The expression levels of beclin 1, AKT, ERK, the phosphorylation of AKT and ERK protein and the ratio of LC3- Ⅱ to LC3- I were detected by Western blotting. Results After 100 MOI AdS-KAI1 infections for 24 h, the rate of cell expressing KAI1 protein reached （ 84.97 ± 8.56） %, number of LC3 increased from 4 to 20 ; and swelling, degeneration of mitochondria was observed, and bilayer-like structure in cytoplasm was found. The expression of beclinl increased （ 1.4 ± 0.3 ） folds, and the expression of LC3- II/LC3- I increased （ 8.00 ± 2.78） folds. PI3K blockade LY294002 pretreatment significantly suppressed the phosphorylation of AKT of MiaPaCa-2 （2. 756 vs 1. 516）, but it did not inhibit the increase of ratio of LC3-Ⅱ to LC3-I （0. 770 vs 1. 403）. ERK blockade PD98059 pretreatment not only significantly suppressed the phosphocylation of ERK of MiaPaCa-2 （1. 637 vs 0. 403 ） , but also inhibit the up-regulation of beclin 1 protein expression （2. 377 vs 1. 150） and increase of ratio of LC3-lI to LC3- I （2. 225 vs 0. 680）. Conclusions KAI1 can significantly induce autophagy of human pancreatic cell line MiaPaCa-2 through phosphorylation of ERK rather than AKT.

关 键 词：胰腺肿瘤 基因 肿瘤抑制 自噬 KAI1 

分 类 号：R735.9[医药卫生—肿瘤]