肿瘤相关成巨噬细胞外泌体miR-92b靶向PTEN而诱导三阴性乳腺癌细胞自噬  

Tumor-associated macrophage exosomal miR-92b target PTEN to induce autophagy in triple-negative breast cancer cells

作　　者：张竞宇[1] 刘晓丹[1] 范莹[1] 温健[1] ZHANG Jing-yu;LIU Xiao-dan;FAN Ying;WEN Jian(The Fifth Department of General Surgery,The Fourth Affliated Hospital of China Medical University,Shenyang 110032,Liaoning Province,China)

机构地区：[1]中国医科大学附属第四医院第五普通外科,辽宁沈阳110032

出　　处：《中国临床药理学杂志》2023年第1期32-36,共5页The Chinese Journal of Clinical Pharmacology

基　　金：辽宁省教育厅科学技术研究基金资助项目(LK201604)。

摘　　要：目的探究肿瘤相关巨噬细胞外泌体中的微小RNA-92b(miR-92b)对三阴性乳腺癌细胞自噬的影响及机制。方法THP-1被诱导细胞分化为M0、M1、M2型巨噬细胞,提取其所分泌的外泌体以及转染miR-92b mimic、Negative Control的M2型巨噬细胞所分泌的外泌体;将10μg·mL^(-1)的上述外泌体以及等体积的磷酸盐缓冲液与MDA-MB-231共孵育48 h(M0 exo组、M1 exo组、M2 exo组、miR-92b exo组、Negative Control exo组、PBS组),MDA-MB-231细胞转染miR-92b mimic、Negative Control(miR-92b组、Negative Control组),MDA-MB-231细胞与10μg·mL^(-1)miR-92b exo共孵育的同时转染磷酸酶及张力蛋白同源物(PTEN)质粒(miR-92b exo+PTEN组),透射电镜观察上述细胞中的自噬小体,蛋白质印迹法检测上述细胞中自噬蛋白微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)、p62的表达,双荧光素酶报告基因实验验证miR-92b和PTEN的靶向关系。结果PBS组、M0 exo组、M1 exo组、M2 exo组、Negative Control组、miR-92b组、miR-92b exo组和miR-92b exo+PTEN组MDA-MB-231细胞自噬小体数分别为(7.00±1.00),(7.33±2.08),(2.00±1.00),(12.67±1.53),(4.00±1.00),(12.67±2.52),(4.00±1.00)和(13.33±1.53)个;这8组MDA-MB-231细胞LC3-Ⅱ相对蛋白表达水平分别为0.94±0.02,0.95±0.02,0.06±0.01,1.30±0.06,0.48±0.06,1.83±0.04,0.27±0.03和1.13±0.05;这8组MDA-MB-231细胞p62相对蛋白表达水平分别为1.08±0.03,1.10±0.06,2.15±0.09,0.27±0.03,1.56±0.07,0.30±0.04,0.17±0.02和1.78±0.14。上述指标,M1 exo组和M2 exo组与PBS组相比,Negative Control组与miR-92b组相比,miR-92b exo组与miR-92b exo+PTEN组相比,差异均有统计学意义(均P<0.01)。结论外泌体miR-92b可通过抑制PTEN的表达而促进乳腺癌细胞的自噬。Objective To explore the effect and mechanism of microRNA-92b(miR-92b)in tumor-associated macrophage exosomes on autophagy in triple-negative breast cancer cells.Methods THP-1 was induced to differentiate into M0,M1,and M2macrophages,and exosomes secreted by these cells and M2 macrophages transfected with miR-92b mimic and Negative Control were extracted.The above exosomes(10μg·mL^(-1))and an equal volume of phosphate buffer solution was incubated with MDA-MB-231 for 48 h(M0 exo group,M1 exo group,M2 exo group,miR-92b exo group,Negative Control exo group,PBS group).MDA-MB-231 cells were transfected with miR-193a-3p mimic,Negative Control(miR-92b group,Negative Control group).MDA-MB-231 cells were incubated with 10μg·mL^(-1)miR-92b exo and simultaneously transfected with phosphatase and tensin homolog(PTEN)plasmid(miR-92b exo+PTEN Group).Transmission electron microscope was used to observe autophagosomes in the above cells,Western blot was used to detect the expression of autophagy protein microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ),p62 in the above cells,dual luciferase reporter gene experiment to verify the targeting relationship between miR-92b and PTEN.Results The number of autophagosomes in MDA-MB-231 cells in PBS group,M0 exo group,M1 exo group,M2 exo group,negative control group,miR-92b group,miR-92b exo group and miR-92b exo+PTEN group was 7.00±1.00,7.33±2.08,2.00±1.00,12.67±1.53,4.00±1.00,12.67±2.52,4.00±1.00 and13.33±1.53,respectively.The relative protein expression levels of LC3-Ⅱin these 8 groups of MDA-MB-231cells were 0.94±0.02,0.95±0.02,0.06±0.01,1.3±0.06,0.48±0.06,1.83±0.04,0.27±0.03 and1.13±0.05,respectively.The relative protein expression levels of p62 in these eight groups of MDA-MB-231 cells were 1.08±0.03,1.10±0.06,2.15±0.09,0.27±0.03,1.56±0.07,0.30±0.04,0.17±0.02 and1.78±0.14,respectively,and the differences of the above indexes were statistically significant(all P<0.01).Conclusion MiR-92b in tumor-associated macrophage exosomes can promote autoph

关 键 词：肿瘤相关巨噬细胞 微小RNA-92b 三阴性乳腺癌 自噬 

分 类 号：R97[医药卫生—药品]