黄芪甲苷调控外泌体介导的JMJD3/H3K27me3/OPN通路抗结直肠癌肝转移机制研究  

Mechanism of AstragalosideⅣregulates extracellular vesicles‑mediated JMJD3/H3K27me3/OPN pathway against colorectal cancer liver metastasis

作　　者：李玲[1] 季青[1] 周晶 LI Ling;JI Qing;ZHOU Jing(Department of Medical Oncology&Cancer Institute of Integrative Medicine,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学附属曙光医院肿瘤科/肿瘤研究室,上海201203

出　　处：《上海中医药杂志》2023年第11期42-49,共8页Shanghai Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(82274297,82074225);中国博士后基金面上项目(2022M722159);上海市炎癌转化病证生物学前沿研究基地项目(2021KJ03-12)。

摘　　要：目的研究黄芪甲苷(ASⅣ)抑制结直肠癌肝转移的作用机制,探索ASⅣ对外泌体介导的Jumonji结构域包含蛋白3(JMJD3)/第27位的赖氨酸三甲基化的组蛋白H3(H3K27me3)/骨桥蛋白(OPN)信号通路的影响。方法取对数生长期的MC38细胞,超速离心法提取MC38细胞外泌体(MC38-EVs)及ASⅣ干预后MC38细胞外泌体(ASⅣ/MC38-EVs),粒径鉴定后用于动物实验。选取C57BL/6J小鼠15只,建立结直肠癌原位肝转移模型后,按照随机数字表法分为模型组、MC38-EVs组、ASⅣ/MC38-EVs组,每组5只。模型组造模前1周尾静脉注射磷酸盐缓冲液(PBS),100μL/次,隔日1次,连续3周;MC38-EVs组和ASⅣ/MC38-EVs组造模前1周尾静脉分别注射0.1 g/L的MC38-EVs、ASⅣ/MC38-EVs,100μL/次,隔日1次,连续3周。4周后处死小鼠,观察结直肠癌肝转移情况;苏木精-伊红(HE)染色法观察肝转移灶病理学改变;蛋白质免疫印迹(Western blot)法检测肝转移灶中JMJD3、H3K27me3、OPN蛋白表达情况;免疫荧光法检测肝转移灶中F4/80+、CD206+巨噬细胞的浸润和JMJD3、H3K27me3、OPN蛋白表达;实时荧光定量逆转录聚合酶链式反应(RT-qPCR)检测瘤体中JMJD3、OPN mRNA表达。结果与模型组比较,MC38-EVs组肝转移灶数目和肝脏质量明显增加(P<0.05);与MC38-EVs组比较,ASⅣ/MC38-EVs组肝转移灶数目和肝脏质量明显减少(P<0.05);免疫荧光结果显示,与模型组比较,MC38-EVs组中肝组织F4/80+、CD206+巨噬细胞浸润程度明显升高(P<0.05);与MC38-EVs组比较,ASⅣ/MC38-EVs组中肝组织巨噬细胞浸润程度明显降低(P<0.05);Western blot、RT-qPCR和免疫荧光结果显示,与模型组比较,MC38-EVs组JMJD3、OPN蛋白和mRNA表达水平明显升高(P<0.05),H3K27me3蛋白表达水平明显降低(P<0.05);与MC38-EVs组相比,ASⅣ/MC38-EVs组JMJD3、OPN蛋白和mRNA表达水平明显降低(P<0.05),H3K27me3蛋白表达水平明显升高(P<0.05)。结论ASⅣ能够有效抑制结直肠癌肝转移,其机制与调控外泌体介导的JObjective To explore the mechanism of AstragalosideⅣ(ASⅣ)in inhibiting colorectal cancer liver metastasis and to investigate the effect of ASⅣon extracellular vesicle-mediated Jumonji domain-containing protein-3(JMJD3)/trimethylated histone H3 at lysine 27(H3K27me3)/osteopontin(OPN)signaling pathway.Methods MC38 cells at the logarithmic growth stage were taken,and MC38 cell extracellular vesicles(MC38-EVs)and MC38 cell extracellular vesicles after ASⅣintervention(ASⅣ/MC38-EVs)were extracted by ultracentrifugation,and the particle size was identified and used for animal experiments.After establishing the orthotopic liver metastasis model of colorectal cancer,fifteen C57BL/6J mice were randomly divided into model group,MC38-EVs group and ASⅣ/MC38-EVs group,with 5 animals in each group.The model group was injected with PBS in the tail vein 1 week before modeling,100μL per time,every other day for 3 weeks;the MC38-EVs group and the ASⅣ/MC38-EVs group were injected with 0.1 g/L,100μL per time MC38-EVs and ASⅣ/MC38-EVs in the tail vein 1 week before modeling respectively,every other day for 3 weeks.The mice were executed after 4 weeks to observe the liver metastases of colorectal cancer;HE staining was performed to observe the pathological changes of liver metastases;Western blot was performed to detect the expression of JMJD3,H3K27me3 and OPN in liver metastases;immunofluorescence was performed to detect the infiltration of F4/80+,CD206+macrophages and the expression of JMJD3,H3K27me3,and OPN in liver metastases;RT-qPCR detected JMJD3,OPN mRNA expression in the tumor.Results The number of metastases and weight of liver were significantly higher in the MC38-EVs group compared with the model group(P<0.05),while those were significantly lower in the ASⅣ/MC38-EVs group compared with the MC38-EVs group(P<0.05);the immunofluorescence results showed that the extent of F4/80+,CD206+macrophage infiltration in liver tissue was significantly higher in the MC38-EVs group compared with the model group(P<0.05),b

关 键 词：结直肠癌 肿瘤转移 黄芪甲苷 外泌体 肿瘤相关巨噬细胞 中药 药理作用 

分 类 号：R285.5[医药卫生—中药学]