HMGA1对TGF-β1诱导HTR-8/SVneo细胞上皮间质转化的影响  

Effects of HMGA1 on TGF-β1-induced epithelial mesenchymal transformation in HTR-8/SVneo cells

作　　者：刘昕 万俊 肖笛鸣 涂玲[1] 魏明[1] LIU Xin;WAN Jun;XIAO Diming(Department of Hematology,The Fifth Affiliated Hospital of Zhengzhou University,Henan,Zhengzhou 450052,China;不详)

机构地区：[1]郑州大学第五附属医院输血科,郑州市450052 [2]郑州大学第五附属医院全科医学科,郑州市450052

出　　处：《河北医药》2023年第5期657-661,666,共6页Hebei Medical Journal

基　　金：河南省医学科技攻关项目资助(编号:LHGJ 20190419)。

摘　　要：目的研究高迁移率蛋白A1(HMGA1)对HTR-8/SVneo细胞中转化生长因子β1(TGF-β1)诱导所致上皮间质转化(EMT)过程中的作用。方法将HMGA1的小分子干扰RNA(siRNA)转染进人绒毛膜滋养层细胞HTR-8/SVneo,将细胞分为空白对照组、阴性对照+lipofectamine 3000组、lipofectamine 3000组、阳性对照+lipofectamine 3000组、HMGA1 SiRNA-1+lipofectamine 3000组、HMG1 SiRNA-2+lipofectamine 3000组、HMG1 siRNA-3+lipofectamine 3000组,采用实时荧光定量PCR(RT-qPCR)和蛋白免疫印迹法,检测各组的HMGA1基因和蛋白表达,筛选出沉默效率最佳的siRNA;TGF-β1诱导HTR-8/SVneo发生EMT,研究TGF-β1对EMT过程的影响;使用筛选后的siRNA与TGF-β1共同作用,检测HMGA1、N-cadherin、E-cadherin的表达情况;加入TGF-β1用于诱导细胞,探究PI3K/AKT和NF-κB通路相关蛋白的表达状况;利用PI3K/AKT和NF-κB通路抑制剂阻断通路,研究HMGA1在通路介导下EMT过程的作用机制。结果HMGA1 siRNA-1组沉默效果最佳,可用于后续实验;与空白对照组相比,TGF-β1组E-cadherin蛋白表达水平降低,N-cadherin蛋白表达水平升高,差异有统计学意义(P<0.05);干扰HMGA1表达可以阻止TGF-β1诱导HTR-8/SVneo细胞发生EMT;TGF-β1诱导后,通路相关蛋白p-AKT、AKT、NF-κB表达量均增加,与空白对照组相比,差异有统计学意义(P<0.05);加入通路抑制剂后,HMGA1的表达量主要受PI3K/AKT通路的影响,而NF-κB通路的抑制剂对HMGA1的影响较小。结论HMGA1在TGF-β1诱导的HTR-8/SVneo细胞EMT过程中发挥着重要作用,而且TGF-β1主要通过PI3K/AKT通路调控HMGA1的表达,进而促进细胞发生EMT。Objective To study effects of high mobility protein A1(HMGA1)on TGFβ1-induced epithelial mesenchymal transformation(EMT)in HTR-8/SVneo cells.Methods HTR 8/SVneo was transfected with small interfering RNA(siRNA)of HMGA1 and entered Human villous trophoblasts(HTR8/SVneo),the cells were divided into the blank control group,negative control+lipofectamine 3000 group,lipofectamine 3000 group,positive control+lipofectamine 3000 group,HMGA1 SiRNA-1+lipofectamine 3000 group,HMG1 SiRNA 2+lipofectamine 3000 group,and HMG1 siRNA 3+lipofectamine 3000 group,the expressions of HMGA1 gene and protein in groups were detected by real-time fluorescence quantitative PCR(RT qPCR)and Western blot,and the siRNA with the best silencing efficiency was screened.TGF-βinduced HTR 8/SVneo to generate EMT,and impacts of TGF-β1 on EMT process.The combined action of siRNA and TGFβscreened was used to detect the expressions of HMGA1,N cadherin and E cadherin.TGFβwas applied to induce cells,and the expressions of PI3K/AKT and NF-κB pathway related proteins were explored.PI3K/AKT and NF-κB pathway inhibitor blocked the pathway,the mechanism of HMGA1 in the pathway mediated EMT process was studied.Results The HMGA1 siRNA-1 was found to have the best silencing efficacy in groups,and it was eligible to be used in the following experiments.Knockdown of HMGA1 significantly down regulated E-cadherin and upregulated N-cadherin(P<0.05).Compared with the blank control group,the expression level of E cadherin protein in the TGF-βgroup decreased,and the expression level of N cadherin protein increased(P<0.05);Interference on the expression of HMGA1 could prevent TGF-β1 from inducing EMT in HTR 8/SVneo cells.The expression level of pathway-related proteins AKT,AKT and NF-κb after being induced with TGF-βincreased significantly when compared with the blank control group(P<0.05).The expression of HMGA1 was mainly impacted by PI3K/AKT pathway after being added with the pathway inhibitor,while NF-κinhibitors of B pathway had insignificant impacts on H

关 键 词：高迁移率族蛋白A1 HTR-8/SVneo:TGF-β1 上皮间质转化 

分 类 号：R329.21[医药卫生—人体解剖和组织胚胎学]