SFRP1基因沉默抑制人胃黏膜上皮GES-1细胞失巢凋亡  

Silence of SFRP1 inhibits anoikis in human gastric mucosal epithelial cell line GES-1

作　　者：汤晓琳[1] 杨丹[2] 吕鑫 沈薇[4] TANG Xiao-Lin;YANG Dan;LüXin(Department of Functional Experiment Center,Shenyang Medical College,Shenyang 110034,Liaoning,China)

机构地区：[1]沈阳医学院机能实验中心,辽宁沈阳110034 [2]沈阳医学院药理教研室 [3]沈阳医学院 [4]沈阳医学院病理生理教研室

出　　处：《中国老年学杂志》2019年第16期4034-4038,共5页Chinese Journal of Gerontology

基　　金：国家自然科学基金资助项目(31501125)

摘　　要：目的观察RNA干扰沉默分泌型卷曲相关蛋白(SFRP)1基因对人胃黏膜上皮细胞(GES)-1失巢凋亡的影响,并探讨其分子机制。方法应用SFRP1基因小干扰RNA(siRNA)转染处理GES-1后,分别采用Realtime RT-PCR和Western印迹检测SFRP1 mRNA和蛋白表达。细胞悬浮培养应用Poly-HEMA方法。采用流式细胞术和Calcein AM/EthD-1荧光双染法检测细胞失巢凋亡,软琼脂集落实验检测细胞非锚定生长状态,免疫荧光和Western印迹实验检测SFRP1沉默后Wnt通路中β-catenin的定位及表达,Realtime RT-PCR检测β-catenin靶基因c-Myc和CyclinD1 mRNA表达。结果与对照组比较,siRNA-891组中SFRP1 mRNA和蛋白表达明显被抑制。siRNA-891组细胞失巢凋亡率明显下降(P<0.05),EthD-1荧光染色的失巢凋亡细胞数目明显减少(P<0.05),软琼脂集落形成数目明显增多(P<0.05)。siRNA-891组中β-catenin蛋白表达水平明显增加,其靶基因c-Myc mRNA和CyclinD1 mRNA表达明显增高(P<0.01)。结论SFRP1siRNA可抑制GES-1细胞失巢凋亡,使其失巢凋亡敏感性下降,激活Wnt分子通路可能是其机制之一。Objective To explore the effect of SFRP1 small interfering RNA(siRNA)on anoikis of human gastric mucosal epithelial cell line GES-1 and the underlying molecular mechanism.Methods SFRP1 siRNA was transfected into GES-1 cells.The SFRP1 mRNA and protein expressions were detected by realtime RT-PCR and Western blot respectively.The experiment of suspension culture was used by Poly-HEMA culture system.Anoikis detections were conducted by anoikis assay kit and flow cytometry.The anchorage-independent growth of cells was determined by assaying colony formation in soft agar.Fluorescent immunocytochemistry and Western blot were used to detect the localization and expression ofβ-catenin in Wnt pathway after SFRP1 silencing.β-catenin target genes c-Myc and CyclinD1 mRNA expressions were measured by realtime RT-PCR.Results Compared with those of control group,SFRP1 mRNA and protein expressions were significantly inhibited in SFRP1 siRNA group.The anoikis rate of SFRP1 siRNA group cells was significantly decreased(P<0.05)and anoikis cells with EthD-1 fluorescent dye were significantly decreased(P<0.05).The colonies in SFRP1 siRNA group were significantly increased(P<0.05).β-catenin protein expression and its target genes c-Myc and CyclinD1 mRNA expressions were significantly increased in SFRP1 siRNA group(P<0.01).Conclusions Interference of SFRP1 inhibits anoikis sensitivity of GES-1 cells.The activation of Wnt pathway might be one of its mechanism.

关 键 词：分泌型卷曲相关蛋白(SFRP)1 失巢凋亡 RNA干扰 Wnt/β-catenin信号通路 

分 类 号：R363.2[医药卫生—病理学]