ANKRD49通过增加Snail/Slug/ZEB1表达促进NCI-H1299细胞上皮-间充质转化的研究  

ANKRD49 promoted epithelial-mesenchymal transition by increasing expression of Snail/Slug/ZEB1 in NCI-H1299 cells

作　　者：高睿 刘超锋[1] 胡金瑞 梁刚 付荣 王维[2] 王海龙[2] 庞敏[1] GAO Rui;LIU Chaofeng;HU Jinrui;LIANG Gang;FU Rong;WANG Wei;WANG Hailong;PANG Min(Department of Pulmonary and Critical Care Medicine,the First Hospital,Shanxi Medical University,Shanxi Province Key Laboratory of Respiratory Disease,Taiyuan 030001,China;Basic Medical Science Center,Institute of Oncobiology,Shanxi Medical University,Jinzhong 030600,China;Department of Pathology,the First Hospital,Shanxi Medical Uni-versity,Taiyuan 030001,China)

机构地区：[1]山西医科大学第一医院呼吸与危重症医学科,呼吸疾病防治与基础研究山西省重点实验室,山西太原030001 [2]山西医科大学基础医学研究中心,肿瘤生物学研究所,山西晋中030600 [3]山西医科大学第一医院病理科,山西太原030001

出　　处：《中国病理生理杂志》2024年第7期1190-1196,共7页Chinese Journal of Pathophysiology

基　　金：山西省自然科学基金资助项目(No.202203021212041)。

摘　　要：目的:研究锚蛋白重复结构域蛋白49(ANKRD49)对人肺腺癌NCI-H1299细胞上皮-间充质转化(EMT)的影响及其机制。方法:构建ANKRD49过表达的NCI-H1299肺腺癌细胞株,显微镜下观察ANKRD49过表达下细胞形态变化;采用RT-qPCR和Western blot法检测EMT相关指标[上皮钙黏素(E-cadherin)、转化生长因子β1(TGF-β1)、波形蛋白(vimentin)、α-平滑肌肌动蛋白(α-SMA)]及EMT相关转录因子(Snail、Slug、Twist与ZEB1)的mRNA和蛋白表达水平;采用细胞免疫荧光观察E-cadherin和vimentin在细胞中的定位和表达水平;免疫组织化学法检测ANKRD49过表达的H1299细胞及对照细胞接种裸鼠肺组织E-cadherin、α-SMA、Snail、Slug及ZEB1的表达。结果:与对照组相比,过表达ANKRD49组细胞表现出间充质细胞样形态(梭形样且紧密连接减少);RT-qPCR和Western blot结果显示ANKRD49过表达组间充质标志物vimentin和α-SMA的mRNA和蛋白质水平显著高于对照组,上皮标志物E-cadherin的mRNA和蛋白质水平低于对照组;与对照组相比,ANKRD49过表达后E-cadherin免疫荧光强度减弱,而vimentin免疫荧光强度显著增加;与对照组相比,ANKRD49过表达后显著增加Snail、Slug与ZEB1的表达;与对照组相比,ANKRD49过表达的NCI-H1299细胞接种裸鼠肺组织中E-cadherin显著减少,α-SMA、Snail、Slug及ZEB1的表达显著增加。结论:ANKRD49通过增加Snail1、Slug与ZEB1的表达来抑制E-cadherin的表达,提高vimentin和α-SMA的表达进而促进NCI-H1299细胞EMT发生。AIM:To investigate the effect of ankyrin repeat domain 49(ANKRD49)on epithelial-mesenchymal transition(EMT)in NCI-H1299 cells,and to explore its mechanism.METHODS:The ANKRD49 was over-expressed in NCI-H1299 cells.The morphological changes of ANKRD49-overpressing NCI-H1299 cells were observed under microscope.The mRNA and protein expression levels of EMT-related markers[E-cadherin,transforming growth factor-β1(TGF-β1),vimentin andα-smooth muscle actin(α-SMA)]and EMT-related transcription factors(Snail1,Slug,Twist and ZEB1)were detected by RT-qPCR Western blot.Immunofluorescence staining was performed to observe the localization and expression of E-cadherin and vimentin in ANKRD49-overexpressing cells or control cells.Immunohistochemical method was performed to examine the levels of E-cadherin,α-SMA,Snail,Slug and ZEB1 in lung tissues of nude mice inoculated with ANKRD49-overexpressing H1299 cells or control cells.RESULTS:Compared with the control group,the ANKRD49 overexpressing cells showed mesenchymal cell morphology(fusiform and less tight connections).RT-qPCR and Western blot results showed that the mRNA and protein levels of mesenchymal markers vimentin andα-SMA in ANKRD49 overexpressing cells were significantly higher than those in cells of control group,while the mRNA and protein levels of epithelial marker E-cadherin were lower than those in cells of control group.Compared with control group,the immunofluorescence intensity of E-cadherin of H1299 cells decreased in after ANKRD49 overexpression,while that of vimentin increased significantly.Snail,Slug and ZEB1 expression were significantly elevated in ANKRD49 overexpressing cells compared with control group.The levels of E-cadherin in lung tissues of nude mice inoculated with ANKRD49-overexpressing H1299 cells declined,while the levels ofα-SMA,Snail,Slug and ZEB1 increased compared with those in control mice.CONCLUSION:ANKRD49 promoted EMT of NCI-H1299 cells by increasing the expression of Snail1,Slug and ZEB1 and consequent downreguation of E-cadher

关 键 词：锚蛋白重复结构域蛋白49 肺腺癌 上皮-间充质转化 

分 类 号：R363.2[医药卫生—病理学] R730.23[医药卫生—基础医学] R734.2