MKK6过表达调控SIRT1影响食管癌细胞肿瘤行为学的体外研究  

Study of Mechanism of Esophageal Cancer Development through MKK6 Over-expression Regulated SIRT1 Expression Level

作　　者：杨晓龙[1] 胡开锋 刘明佳[1] 任朋亮[1] 陈利弘[1] 刘戟[1] 

机构地区：[1]四川大学华西基础医学与法医学院生物化学与分子生物学教研室,成都610041

出　　处：《四川大学学报（医学版）》2015年第4期548-553,共6页Journal of Sichuan University(Medical Sciences)

摘　　要：目的向食管癌Eca109细胞中转染pcDNA3.1(+)/myc-His A-丝裂原活化蛋白激酶6(MKK6)重组过表达载体,观察过表达MKK6影响沉默信息转录调控因子(SIRT1)表达后肿瘤细胞增殖、凋亡以及侵袭力发生的变化,探讨p38-MAPK-SIRT1调节轴的存在性及其对Eca109细胞各项肿瘤行为学造成的影响。方法根据GenBank中MKK6基因的mRNA序列设计引物,构建MKK6过表达载体;将Eca109细胞分空载体转染组、MKK6过表达载体转染组、MKK6-SIRT1ShRNA共转染组和MKK6-RES(白藜芦醇)组,相应处理后,采用Western blot测定各组Eca109细胞的SIRT1及MKK6表达水平,MTT法检测各组Eca109细胞的增殖活力,Transwell法观察各组Eca109细胞的侵袭力,流式细胞术检测各组Eca109细胞的凋亡情况。结果测序结果表明成功构建MKK6过表达载体;转染MKK6过表达载体可降低Eca109细胞的内源性SIRT1表达,减弱Eca109细胞的增殖活力,促其凋亡,同时可使Eca109细胞的侵袭力增强,以上各项改变均与SIRT1的表达水平有关。结论Eca109细胞内可能存在p38-MAPK-SIRT1调节轴,MKK6及其下游因子对SIRT1可能具有反馈调节作用,可引起Eca109细胞各项肿瘤行为学的变化。Objective To observe the influence of silent information regulator of transcription 1（SIRT1）level by regulation of MKK6over-expression in esophageal cancer cell Eca109,and then to explore the relationship between p38-MAPK-SIRT1 axis and progressing of esophageal cancer.Methods MKK6over-expression vector was successfully constructed firstly.Then the divided Eca109 cells were treated according to four groups：empty vector group,MKK6over-xpression group（MKK6group）,MKK6-SIRT1 ShRNA group,MKK6-RES group.The expression of MKK6 and endogenous SIRT1 were tested by Western blot;cell proliferation capability was detected by MTT method;cell invasion force was observed by transwell method;and cell apoptosis was detected with flow cytometry.Results pcDNA3.1（＋）/myc-His A-MKK6 over-expression vector was constructed successfully and proved by sequencing.MKK6＇s over-expressing could reduce the expression of endogenous SIRT1.The viability of Eca109 cells was decreased.The increasing of invasion and apoptosis was observed.Conclusion There might be the p38-MAPK-SIRT1 regulation axis in Eca109 cells and affecting on a series of physiological characteristics of Eca109 cells.

关 键 词：MKK6 SIRT1 ECA109细胞 细胞活力 细胞侵袭力 细胞凋亡 反馈调节轴 

分 类 号：R735.1[医药卫生—肿瘤]