肿瘤源性IL-1β激活血管内皮细胞中NF-κB p65通路促进其对肝癌细胞的粘附  

Tumor-Derived IL-1β Promotes Adhesion of Vascular Endothelial Cells with Hepatoma Cells Through NF-κB p65 Signaling Pathway

作　　者：房佩佩 付晓燕[1] 吴沛恩 吴圆圆 张倩[1] 张凯铖 彭美玉 梁淑娟[1] Fang Peipei;Fu Xiaoyan;Wu Peien(Shandong Provincial University Key Laboratory of Immunology,Department of Immunology,Weifang Medical University,Weifang 261053,China)

机构地区：[1]潍坊医学院免疫学教研室,山东省高校免疫学重点实验室,潍坊261053

出　　处：《华中科技大学学报（医学版）》2022年第3期294-302,共9页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.81972695)。

摘　　要：目的 探讨肿瘤来源的白细胞介素1β(IL-1β)对血管内皮细胞粘附肝癌细胞功能的影响及分子机制。方法 慢病毒转染建立稳定表达IL-1β(HepG2/IL-1β)和GFP(HepG2/GFP)的HepG2细胞,ELISA法测定细胞培养上清中IL-1β浓度,以含IL-1β的HepG2细胞条件培养液(IL-1β-CM)作为肿瘤源性IL-1β来源,以GFP慢病毒感染的HepG2细胞条件性培养液(Ctrl-CM)为对照。以含10 ng/mL IL-1β的IL-1β-CM刺激人脐静脉内皮细胞(HUVECs+IL-1β-CM),观察其对HepG2/IL-1β、HepG2/GFP细胞的粘附能力,并与HUVECs+Ctrl-CM比较。qRT-PCR法检测血管内皮细胞中E-选择素(CD62E)、细胞间粘附分子-1(ICAM-1,即CD54)、血管细胞粘附分子-1(VCAM-1,即CD106)mRNA表达水平。流式细胞术检测CD62E、CD54蛋白表达水平。Western blot分析HUVECs中IL-1β-CM激活的信号通路,以及靶向阻断信号通路对粘附分子表达和细胞间粘附作用的影响。结果 肿瘤源性IL-1β显著促进了HUVECs对HepG2细胞的粘附作用,明显上调HUVECs细胞中CD62E、CD54 mRNA表达水平。CD62E蛋白在IL-1β刺激后4 h短暂升高,CD54蛋白呈现为持续升高模式,自刺激4 h起维持至24 h。IL-1β激活血管内皮细胞中NF-κB p65、p38 MAPK信号通路,其中NF-κB p65位于上游且对p38 MAPK活化具有控制作用。靶向阻断NF-κB p65通路显著下调了HUVECs表面CD62E、CD54表达和对HepG2细胞的粘附效应。结论 肿瘤源性IL-1β通过激活NF-κB p65为主的信号机制调控血管内皮细胞CD62E和CD54表达,促进血管内皮细胞对肝癌细胞的粘附作用,进而在肝癌细胞的浸润转移中发挥作用。Objective To elucidate the effect of tumor-derived interleukin-1 beta(IL-1β)on adhesion of vascular endothelial cells(HUVECs)with hepatoma cells and the potential molecular mechanisms.Methods HepG2 cell line stably expressing IL-1β(HepG2/IL-1β)and GFP(HepG2/GFP)was established by lentivirus transfection, and the IL-1β in cell culture supernatant was determined by ELISA.Conditioned medium(CM)containing IL-1β(IL-1β-CM)of HepG2 cells was used as a source for tumor-derived IL-1β,and was controlled with CM from HepG2/GFP cells(Ctrl-CM).HUVECs stimulated with 10 ng/mL IL-1β(HUVECs+IL-1β-CM)were used to observe their adhesion ability with HepG2/IL-1β and HepG2/GFP cells, and HUVECs+Ctrl-CM served as control.The mRNA expression levels of E-selectin(CD62 E),ICAM-1(CD54)and VCAM-1(CD106)in HUVECs were detected by qRT-PCR.Cell surface expression levels of CD62 E and CD54 were detected by flow cytometry.IL-1β induced signaling pathways in HUVECs were analyzed through Western blotting.Specific inhibitors were used to block each signaling pathway, and the expression of adhesion molecules as well as the adhesion of vascular endothelial cells with hepatoma cells were subsequently monitored.Results Tumor-derived IL-1β significantly enhanced the adhesion of HUVECs with HepG2 cells and upregulated the expression levels of CD62 E and CD54 mRNA in HUVECs cells.The surface CD62 E was temporarily upregulated 4 h after IL-1β stimulation, while the expression of CD54 protein showed a continuous upregulated pattern, which lasted from 4 h to 24 h.IL-1β mainly activated NF-κB p65 and p38 MAPK signaling pathways in HUVECs, and NF-κB p65 was proved to be located at upstream, regulating the activation of p38 MAPK pathway.Targeted blocking of NF-κB p65 pathway by specific inhibitor significantly downregulated the expression of CD62 E and CD54 on surface of HUVECs and inhibited their adhesion ability with HepG2 cells.Conclusion Tumor-derived IL-1β promotes adhesion of HUVECs with hepatoma cells through upregulation of CD62 E an

关 键 词：肝癌 白细胞介素1Β 血管内皮细胞 粘附分子 信号通路 

分 类 号：R735.7[医药卫生—肿瘤]