靶向沉默脂肪酸合酶FASN基因对食管癌细胞失巢凋亡及转移水平的调控作用  

Effects of targeted silencing fatty acid synthase FASN gene on apoptosis and metastasis level of esophageal cancer cells in lost nests

作　　者：余方方 李巨元 黄健 吕琼秀 YU Fangfang;LI Juyuan;HUANG Jian;LYU Qiongxiu(Laboratory Department,Hainan Western Central Hospital,Hainan Danzhou 571700,China;Department of Oncology,Hainan Western Central Hospital,Hainan Danzhou 571700,China)

机构地区：[1]海南西部中心医院检验科,海南儋州571700 [2]海南西部中心医院肿瘤科,海南儋州571700

出　　处：《现代肿瘤医学》2023年第15期2809-2815,共7页Journal of Modern Oncology

基　　金：海南省卫生健康行业科研项目(编号:22A200041)。

摘　　要：目的:探究沉默脂肪酸合成酶(FASN)对食管癌肿瘤细胞失巢凋亡及转移的影响。方法:实时荧光定量PCR(qRT-PCR)测定人正常食管上皮细胞系(HEEC)与人食管癌细胞系(CaES-17、EC109、EC9706、KYSE170、TE-1)中FASN mRNA表达水平。实验分为Control组、NC-shRNA组、FASN-shRNA1组、FASN-shRNA2组,分别将慢病毒介导的NC-shRNA、FASN-shRNA1、FASN-shRNA2转染到EC109细胞,倒置显微镜观察绿色荧光表达、qRT-PCR和蛋白质免疫印迹(Western blot)检测转染效果。应用聚羟乙基异丁烯酸(poly-HEMA)包被培养板模拟各组EC109细胞失巢凋亡,AnnexinV-FITC/PI双染法检测细胞凋亡情况,Calcein AM/EthD-1荧光双染法鉴定活细胞与死细胞。免疫荧光染色观察各组EC109细胞内上皮型钙黏素(E-cadherin)、波形蛋白(Vimentin)荧光染色,Western blot测定各组EC109细胞内E-cadherin、Vimentin、锌指转录因子(Snail)、基质金属蛋白酶2(MMP-2)、MMP-9蛋白表达,Transwell实验检测各组EC109细胞迁移与侵袭。结果:与人正常食管上皮细胞系HEEC比较,人食管癌细胞系CaES-17、EC109、EC9706、KYSE170、TE-1中FASN mRNA相对表达量升高(P<0.05)。NC-shRNA组、FASN-shRNA1组与FASN-shRNA2组中绿色荧光表达明显,与NC-shRNA组比较,FASN-shRNA1组与FASN-shRNA2组中FASN mRNA相对表达量与蛋白相对表达量升高(P<0.05),细胞凋亡率增加(P<0.05),EthD-1标记的细胞阳性率也增加(P<0.05),细胞内E-cadherin荧光染色强度增强、Vimentin荧光染色强度减弱,E-cadherin蛋白相对表达量上调(P<0.05),Vimentin、Snail、MMP-2以及MMP-9的蛋白相对表达量均下调(P<0.05),细胞迁移数目与侵袭数目也减少(P<0.05)。结论:慢病毒介导的FASN-shRNA靶向沉默FASN的表达,该作用能够促进食管癌肿瘤细胞失巢凋亡,抑制肿瘤细胞上皮间质转化诱导的迁移与侵袭。Objective:To investigate the effects of silencing fatty acid synthase(FASN)on apoptosis and metastasis of esophageal cancer cells.Methods:Real-time quantitative fluorescent PCR(qRT-PCR)was used to detect FASN mRNA expression levels in human normal esophageal epithelial cell lines(HEEC)and human esophageal cancer cell lines(CaES-17,EC109,EC9706,KYSE170,TE-1).The experiment was divided into Control group,NC-shRNA group,FASN-SHRNA1 group and FASN-SHRNA2 group.Lentivirus-mediated NC-shRNA,FASN-shRNA1 group and FASN-shRNA2 group were transfected into EC109 cells,respectively.The inverted microscope was used to observe the green fluorescense.qRT-PCR and Western blot were used to detect the transfection effect.The apoptosis of EC109 cells in lost nests was simulated by polyhydroxy-hema(Poly-HEMA)coated culture plate.AnnexinV-FITC/PI double staining was used to detect the apoptosis.Calcein AM/EthD-1 fluorescence double staining was used to identify the living and dead cells.Fluorescence staining of E-cadherin and Vimentin in EC109 cells of each group was observed by immunofluorescence staining.The protein expressions of E-cadherin,Vimentin,Snail,matrix metalloproteinase 2(MMP-2)and MMP-9 in EC109 cells of each group were determined by Western blot.Transwell assay was used to detect the migration and invasion of EC109 cells.Results:Compared with HEEC,the relative expression level of FASN mRNA in human esophageal cancer cell lines CaES-17,EC109,EC9706,KYSE170,TE-1 was increased(P<0.05).The green fluorescence expression was obvious in the NC-shRNA group,the FASN-SHRNA1 group and the FASN-SHRNA2 group,compared with the NC-shRNA group,the relative mRNA expression and protein expression of FASN in the FSN-SHRNA1 group and the FASN-SHRNA2 group were increased(P<0.05),the cell apoptosis rate was increased(P<0.05),the positive rate of EthD-1 labeled cells was also increased(P<0.05),the intensity of intracellular E-cadherin fluorescence staining was increased,the intensity of Vimentin fluorescence staining was decreased,the relati

关 键 词：食管癌 脂肪酸合成酶 失巢凋亡 上皮间质转化 迁移 侵袭 

分 类 号：R735.1[医药卫生—肿瘤]