乳腺癌外泌体miR-222-3p靶向CDKN1B诱导肿瘤相关成纤维细胞的形成  

Exosomal miR-222-3p targeting CDKN1B induces the formation of tumor-associated fibroblasts

作　　者：李新涛 胡珍 陈南征[3] 李朱斌 马少君[5] LI Xin-tao;HU Zhen;CHEN Nan-zheng(Department of Internal Medicine,Shaanxi Cancer Hospital,Xi'an Shaanxi 710061,China;Department of Gynaecology,Xi'an Maternal and Child Health Hospital,Xi'an Shaanxi 710002,China;Department of Thoracic Surgery,First Affiliated Hospital of Xi'an Jiaotong University,Xi'an Shaanxi 710061,China)

机构地区：[1]陕西省肿瘤医院内科,陕西西安710061 [2]西安市妇幼保健院妇科,陕西西安710002 [3]西安交通大学第一附属医院胸外科,陕西西安710061 [4]陕西省肿瘤医院微创介入科,陕西西安710061 [5]陕西省人民医院肿瘤外科,陕西西安710061

出　　处：《临床和实验医学杂志》2021年第20期2144-2148,共5页Journal of Clinical and Experimental Medicine

基　　金：陕西省重点研发计划项目(编号:2020SF-056)。

摘　　要：目的探究乳腺癌外泌体miR-222-3p靶向CDKN1B对肿瘤相关成纤维细胞形成的影响及机制。方法HKF细胞分为MCF7 exo组、MDA-MB-231 exo组、MCF10A exo组和PBS组(HKF细胞分别与10μg/mL的MCF7 exo、MDA-MB-231 exo、MCF10A exo以及等体积的PBS共孵育72 h),miR-NC组、miR-222-3p mimic组和miR-222-3p inhibitor组(HKF细胞分别转染Negative Control、miR-222-3p mimic、miR-222-3p inhibitor),miR-NC exo组和miR-222-3p mimic exo组(HKF细胞分别与10μg/mL的miR-NC exo和miR-222-3p mimic exo共孵育72 h),miR-222-3p mimic exo+CDKN1B组(HKF细胞与miR-222-3p mimic exo共孵育72 h后转染CDKN1B质粒)。提取乳腺癌细胞MCF7和MDA-MB-231、乳腺上皮细胞MCF10A所分泌的外泌体(MCF7 exo、MDA-MB-231 exo和MCF10A exo),实时荧光定量聚合酶链式反应(qRT-PCR)检测上述外泌体中miR-222-3p的表达。蛋白质印迹法检测上述各组HKF细胞中基质金属蛋白酶-9(MMP)-9、MMP-2、MMP-14、成纤维细胞活化蛋白(FAP)、纤连蛋白(fibronectin)、波形纤维蛋白(vimentin)、α-平滑肌肌动蛋白(α-SMA),双荧光素酶报告基因实验检测miR-222-3p和周期蛋白依赖激酶抑制因子1B(CDKN1B)结合。结果与PBS组相比,MCF7 exo组、MDA-MB-231 exo组HKF细胞MMP-9、MMP-2、MMP-14、FAP、fibronectin、vimentin、α-SMA表达显著增加,差异均有统计学意义(P<0.001)。与miR-NC组细胞相比,miR-222-3p mimic组细胞MMP-9、MMP-2、MMP-14、FAP、fibronectin、vimentin、α-SMA表达显著增加,差异均有统计学意义(P<0.001),miR-222-3p inhibitor组细胞上述标志物表达显著降低,差异均有统计学意义(P<0.001)。双荧光素酶报告基因检测结果显示CDKN1B为miR-222-3p的靶基因。MCF7 exo组(4.00±0.31)、MDA-MB-231 exo组(5.94±0.14)中miR-222-3p表达显著高于MCF10A exo组(1.00±0.00),差异有统计学意义(P<0.001)。与miR-NC exo组相比,miR-222-3p mimic exo组HKF细胞MMP-9、MMP-2、MMP-14、FAP、fibronectin、vimentin、α-SMA表达显著增加,差异均有统计学意义(P<0.0Objective To explore the effect and mechanism of breast cancer exosomal miR-222-3p targeting CDKN1B on the formation of tumor-associated fibroblasts.Methods HKF cells were divided into MCF7 exo group,MDA-MB-231 exo group,MCF10A exo group and PBS group(HKF cells were combined with 10μg/mL MCF7 exo,MDA-MB-231 exo,MCF10A exo and equal volume of PBS incubated for 72h),miR-NC group,miR-222-3p mimic group and miR-222-3p inhibitor group(HKF cells were transfected with Negative Control,miR-222-3p mimic,miR-222-3p inhibitor),miR-NC exo group and miR-222-3p mimic exo group(HKF cells were incubated with 10μg/mL miR-NC exo and miR-222-3p mimic exo respectively for 72h),miR-222-3p mimic exo+CDKN1B group(HKF cells were incubated with miR-222-3p mimic exo for 72 hours and then transfected with CDKN1B plasmid),and the exosomes secreted by breast cancer cells MCF7 and MDA-MB-231 and breast epithelial cells MCF10A(MCF7 exo,MDA-MB)-231 exo and MCF10A exo),real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of miR-222-3p in the above exosomes.Western blotting was used to detect matrix metalloproteinase-9(MMP)-9,MMP-2,MMP-14,fibroblast activation protein(FAP),fibronectin,and vimentin in the above groups of HKF cells,α-smooth muscle actin(α-SMA),dual luciferase reporter gene experiment to detect the binding of miR-222-3p and cyclin-dependent kinase inhibitor 1B(CDKN1B).Results Compared with the PBS group,MMP-9,MMP-2,MMP-14,FAP,fibronectin,vimentin,α-SMA expression increased significantly in the MCF7 exo and MDA-MB-231 exo groups,and the differences were statistically significant(P<0.001).Compared with miR-NC group cells,miR-222-3p mimic group cells MMP-9,MMP-2,MMP-14,FAP,fibronectin,vimentin,α-SMA expression were increased significantly,and the differences were statistically significant(P<0.001),and the expression of the above-mentioned markers in miR-222-3p inhibitor group cells significantly were decreased,and the differences were statistically significant(P<0.001).The results

关 键 词：乳腺癌 miR-222-3p 肿瘤相关成纤维细胞 

分 类 号：R737.9[医药卫生—肿瘤]