脂多糖反式激活表皮生长因子受体诱导子宫内膜癌增殖和环氧化酶-2表达

Lipopolysaccharide induces endometrial cancer proliferation and cyclooxygenase-2 expression via transactivation of epidermal growth factor receptor

目的 探讨Toll样受体4(TLR4)和表皮生长因子受体(EGFR)对脂多糖促进人子宫内膜癌RL95-2细胞增殖和环氧化酶2(COX-2)/前列腺素E2(PGE2)信号通路的调节作用。方法 采用噻唑蓝(MTT)比色法,检测不同浓度LPS和5μg/mL LPS处理不同时间后的RL95-2细胞的细胞毒性,选择5μg/mL LPS处理细胞24 h,采用实时荧光定量PCR(qRT-PCR)检测细胞Ki-67 mRNA表达水平。将RL95-2细胞随机分为5组:control组、LPS组、LPS+CD14 mAb组、LPS+TLR4抑制剂(TAK-242)组、LPS+EGFR抑制剂(cetuximab)组。control组:不做任何处理;LPS组:5μg/mL LPS刺激细胞24 h;LPS+CD14 mAb组:5μg/mL LPS和10μg/mL CD14 mAb联合干预24 h;LPS+TAK-242组:5μg/mL LPS和10 nmol/L TAK-242联合干预24 h;LPS+cetuximab组:5μg/mL LPS和10μg/mL cetuximab联合干预24 h。分别采用MTT、qRT-PCR、酶联免疫吸附实验(ELISA)方法检测并分析细胞增殖活性、COX-2 mRNA表达水平、PGE2含量的变化情况。结果 脂多糖以时间和剂量依赖的方式诱导RL95-2细胞增殖,上调Ki-67 mRNA表达,并能够以时间依赖方式上调PGE2表达。与control组相比,LPS+CD14 mAb组、LPS+TAK-242组、LPS+cetuximab组均可完全抑制细胞增殖、下调COX-2 mRNA和PGE2表达。结论 LPS诱导子宫内膜癌细胞增殖,上调COX-2和PGE2表达,TLR4信号和EGFR活化在感染促进的子宫内膜癌进展中发挥关键作用,具有一定临床研究价值。

Objective To investigate the regulatory effects of Toll like receptor 4(TLR4) and epidermal growth factor receptor(EGFR) on the proliferation of human endometrial carcinoma RL95-2 cells and cyclooxygenase-2(COX-2)/prostaglandin E2(PGE2) signal pathway stimulated by lipopolysaccharide.Methods The cytotoxicity of RL95-2 cells treated with different concentrations of LPS and 5 μg/mL LPS for different time was detected by thiazolyl blue(MTT) colorimetric method.The cells were treated with 5 μg/mL LPS for 24 h,and the expression level of Ki-67 mRNA in the cells was detected by real-time quantitative PCR(qRT-PCR).The RL95-2 cells were randomly divided into 5 groups:control group,LPS group,LPS+CD14 mAb group,LPS+TLR4 inhibitor(TAK-242) group,and LPS+EGFR inhibitor(cetuximab) group.The control group did not do any treatment;the LPS group was treated with 5 μg/mL LPS for 24 h;the LPS+CD14 mAb group was treated with 5 μg/mL LPS and 10 μg/mL CD14 mAb for 24 h;the LPS+TAK-242 group was treated with 5 μg/mL LPS and 10 10 nmol/L TAK-242 were treated for 24 hours;LPS+cetuximab group was treated with 5 μg/mL LPS and 10 μg/mL cetuximab for 24 hours.MTT,qRT-PCR and enzyme-linked immunosorbent assay(ELISA) were used to detect the changes of cell proliferation activity,COX-2 mRNA expression level and PGE2 content in each group.Results Lipopolysaccharide induced proliferation of RL95-2 cells andup-regulated Ki-67 mRNA expression in a time-dependent and dose-dependent manner,and up-regulated PGE2 expression in a time-dependent manner.Compared with the control group,LPS+CD14 mAb group,LPS+TAK-242 group and LPS+cetuximab group could completely inhibit cell proliferation and down-regulate COX-2 mRNA and PGE2 expression.Conclusion LPS can induce proliferation of endometrial cancer cells and up-regulate COX-2 and PGE2 expression.TLR4 signal pathway and EGFR activation play key roles in the progression of endometrial cancer promoted by infection,which has certain clinical research value.