幽门螺杆菌脂多糖通过调节MDM2表达对胃上皮细胞恶性转化的影响

Effect of Helicobacter pylori Lipopolysaccharide on Malignant Transformation of Gastric Epithelial Cells by Regulating MDM2 Expression

背景:幽门螺杆菌脂多糖(Hp-LPS)在诱导胃癌发生中起重要作用。目的:探讨Hp-LPS对MDM2基因表达的调节作用,及其对胃上皮细胞恶性转化的影响。方法:分别以Hp-LPS(1μg/mL)和大肠埃希菌(E.coli)-LPS(1μg/mL)刺激人胃上皮细胞株GES-1和胃癌细胞株HGC-27、MKN45 24 h,以蛋白质印迹法检测MDM2表达。构建MDM2启动子区荧光素酶报告基因质粒并转染HGC-27细胞,以Hp-LPS刺激细胞24 h后,检测报告基因相对荧光素酶活性。构建MDM2 RNA干扰质粒并分别转染三株细胞,行流式细胞术细胞凋亡检测和Transwell细胞侵袭实验。建立裸鼠MKN45、HGC-27细胞胃癌异种移植瘤模型并予尾静脉注射siRNA-hMDMA2-3,观察移植瘤生长情况。结果:Hp-LPS可增加MDM2基因启动子转录活性,上调胃上皮细胞和胃癌细胞MDM2蛋白表达。转染siRNA-hMDMA2-3的GES-1、HGC-27、MKN45细胞,细胞凋亡较转染阴性对照siRNA者显著增加(P<0.05),侵袭能力显著减弱(P<0.05)。经siRNA-hMDMA2-3干预的异种移植瘤模型裸鼠,肿瘤生长抑制率显著高于以阴性对照siRNA干预者(P<0.05)。结论:Hp-LPS可能通过诱导MDM2表达引起胃上皮细胞恶性转化,而抑制MDM2表达可促进胃上皮细胞和胃癌细胞凋亡并抑制其侵袭能力,发挥抑制胃上皮细胞恶性转化和胃癌生长的作用。

Background:Helicobacter pylori lipopolysaccharide(Hp-LPS)plays an important role in gastric carcinogenesis.Aims:To investigate the regulatory effect of Hp-LPS on MDM2 gene expression and its impact on malignant transformation of gastric epithelial cells.Methods:After stimulated with Hp-LPS 1μg/mL or Escherichia coli(E.coli)-LPS 1μg/mL for 24 hours,expression of MDM2 in human gastric epithelial cell line(GES-1)and gastric cancer cell line(HGC-27 and MKN45)was detected by Western blotting.Plasmid containing MDM2 promoter luciferase reporter gene was constructed and transfected into HGC-27 cells,and the relative luciferase activity was measured after treated with Hp-LPS for 24 hours.RNA interference plasmid targeting to MDM2 was constructed and transfected into GES-1,HGC-27,and MKN45 cells,then changes in cell apoptosis and invasiveness were analyzed by flow cytometry and Transwell invasion assay.Growth inhibition of MKN45 and HGC-27 xenograft tumors in nude mice was observed after receiving caudal vein injection of siRNA-hMDMA2-3 for silencing MDM2 expression.Results:Hp-LPS enhanced the transcriptional activity of MDM2 promoter and up-regulated MDM2 protein expression in gastric epithelial and gastric cancer cells.In GES-1,HGC-27,and MKN45 cells transfected with siRNA-hMDMA2-3,the cell apoptosis was increased and the cell invasive capacity was reduced as compared with those transfected with negative control siRNA(P<0.05).Furthermore,the tumor inhibition rate of nude mice treated with siRNA-hMDMA2-3 was increased significantly as compared with those treated with negative control siRNA(P<0.05).Conclusions:Hp-LPS can induce malignant transformation of gastric epithelial cells by up-regulating MDM2 expression.Inhibiting MDM2 expression might suppress the malignant transformation and tumor growth by inducing cell apoptosis and reducing cell invasiveness.