摘要
目的:探究胃癌外泌体lncRNA CASC11对M2型巨噬细胞极化的影响及机制。方法:人单核细胞THP-1加入PMA诱导其分化为M0型巨噬细胞,M0型巨噬细胞和胃癌细胞MGC-803中转染空白质粒(Vector组)和lncRNA CASC11过表达质粒(lncRNA CASC11组),qRT-PCR转染后巨噬细胞中i NOS、CD16、CD206、Arg-1、Ym1、TNF-α、IL-1β、TGF-β和IL-10 mRNA的表达,流式细胞术检测CD206^(+)CD14^(+)细胞比例。提取人胃粘膜细胞GES-1、人胃癌细胞株MGC-803、SGC7901、MKN45以及Vector和lncRNA CASC11组MGC803细胞所分泌的外泌体(GES-1 exo、MGC-803 exo、MKN45 exo、SGC7901 exo、Vector exo、CASC11exo),qRT-PCR检测lncRNA CASC11在GES-1、MGC-803、SGC7901、MKN45细胞、转染后的MGC-803细胞及其外泌体中的表达。将PBS、GES-1 exo、MGC-803 exo、MKN45 exo、SGC7901 exo、Vector exo、CASC11exo与M0型巨噬细胞共孵育后,qRT-PCR检测细胞中lncRNA CASC11、i NOS、CD16、CD206、Arg-1、Ym1、TNF-α、IL-1β、TGF-β和IL-10的表达,CD206^(+)CD14^(+)细胞比例,Western blot检测TLR4表达以及NF-κB磷酸化水平。结果:lncRNA CASC11在胃癌细胞及其外泌体中均高表达。相比于Vector组,lncRNA CASC11组巨噬细胞中i NOS、CD16、TNF-α和IL-1β表达显著下调,CD206、Arg-1、Ym1、TGF-β和IL-10表达显著上调,CD206^(+)CD14^(+)细胞比例显著增加。相比于PBS组,MGC-803 exo、MKN45 exo和SGC7901 exo组巨噬细胞中i NOS、CD16、TNF-α和IL-1β表达显著下调,lncRNA CASC11、CD206、Arg-1、Ym1、TGF-β和IL-10表达显著上调,CD206^(+)CD14^(+)细胞比例显著增加,TLR4表达以及NF-κB磷酸化水平均显著减少。相比于Vector exo,CASC11 exo中lncRNA CASC11表达显著增加,相比于Vector exo组细胞,CASC11 exo组巨噬细胞中i NOS、CD16、TNF-α和IL-1β表达显著下调,lncRNA CASC11、CD206、Arg-1、Ym1、TGF-β和IL-10表达显著上调,CD206^(+)CD14^(+)细胞比例显著增加,TLR4表达以及NF-κB磷酸化水平均显著减少。结论:胃癌外泌体lncRNA CASC11抑制TLR4/NF-κB信号通路而诱导M2型巨噬细胞极化。
Objective:To explore the effect and mechanism of gastric cancer exosomal lncRNA CASC11 on the polarization of M2 macrophages.Methods:Human monocyte THP-1 was added to PMA to induce differentiation into M0 macrophages,M0 macrophages and gastric cancer cells MGC-803 were transfected with blank plasmids(Vector group)and lncRNA CASC11 overexpression plasmids(lncRNA CASC11)Group.The expression of iNOS,CD16,CD206,Arg-1,Ym1,TNF-α,IL-1β,TGF-βand IL-10 mRNA in macrophages was detected by qRT-PCR.Proportion of CD206^(+)CD14^(+)cells was detected by flow cytometry.Extract the exosomes secreted by human gastric mucosal cells GES-1,human gastric cancer cell lines MGC-803,SGC7901,MKN45,and MGC803 cells in Vector and lncRNA CASC11 group(GES-1 exo,MGC-803 exo,MKN45 exo,SGC7901 exo,Vector exo,CASC11exo),qRT-PCR was used to detect the expression of lncRNA CASC11 in GES-1,MGC-803,SGC7901,MKN45 cells,transfected MGC-803 cells and their exosomes.M0 macrophages was co-incubated with PBS,GES-1 exo,MGC-803 exo,MKN45 exo,SGC7901 exo,Vector exo,CASC11exo,qRT-PCR was used to detect the expression of lncRNA CASC11,iNOS,CD16,CD206,Arg-1,Ym1,TNF-α,IL-1β,TGF-βand IL-10,and the proportion of CD206^(+)CD14^(+)cells.Western blot was used to detect the expression of TLR4 and the level of NF-κB phosphorylation.Results:lncRNA CASC11 is highly expressed in gastric cancer cells and their exosomes.Compared with the Vector group,in the lncRNA CASC11 group,the expressions of iNOS,CD16,TNF-αand IL-1βwere significantly down-regulated,and the expressions of CD206,Arg-1,Ym1,TGF-βand IL-10 were significantly up-regulated,and the proportion of CD206^(+)CD14^(+)cells has increased significantly.Compared with the PBS group,the expressions of iNOS,CD16,TNF-αand IL-1βin macrophages of MGC-803 exo,MKN45 exo and SGC7901 exo groups were significantly down-regulated,lncRNA CASC11,CD206,Arg-1,Ym1,TGF-βand IL-10 expression was significantly up-regulated,the proportion of CD206^(+)CD14^(+)cells was significantly increased,and TLR4 expression and NF-κB phosphorylation levels were significantly reduced.Compared with Vector exo,the expression of lncRNA CASC11 in CASC11 exo was significantly increased.Compared with the Vector exo group,the expression of iNOS,CD16,TNF-αand IL-1βin the macrophages of the CASC11 exo group was significantly down-regulated,and the expression of lncRNA CASC11,CD206,Arg-1,Ym1,TGF-βand IL-10 were significantly up-regulated,the proportion of CD206^(+)CD14^(+)cells was significantly increased,and the expression of TLR4 and NF-κB phosphorylation levels were significantly reduced.Conclusion:Gastric cancer exosomal lncRNA CASC11 inhibits the TLR4/NF-κB signaling pathway and induces polarization of M2 macrophages.
作者
周安付
谭琰
张惠晶
ZHOU Anfu;TAN Yan(The First Affiliated Hospital of Hainan Medical College,Hainan Haikou 570102,China)
出处
《河北医学》
CAS
2021年第12期1948-1957,共10页
Hebei Medicine
基金
海南省卫生健康行业科研项目,(编号:20A200170)
国家自然科学基金项目,(编号:81860650)。
