MicroRNA-520e通过靶向抑制AEG-1发挥抗结直肠癌特性  

MicroRNA-520e Exerts Anti-colorectal Cancer Properties by Targeting AEG-1

作　　者：吕飒美[1] 张健[1] 吴友伟[1] 刘倩[1] 易默[1] Lü Sa-mei;ZHANG Jian;WU You-wei;LIU Qian;YI Mo(The Second Department of Gastroenterology,Shaanxi Provincial People's Hospital,Xi'an,Shaanxi,710068,China)

机构地区：[1]陕西省人民医院消化内二科,陕西西安710068

出　　处：《现代生物医学进展》2022年第17期3229-3237,共9页Progress in Modern Biomedicine

基　　金：陕西省自然科学基础研究计划项目(2018JM7118)。

摘　　要：目的:探究MicroRNA-520e(miR-520e)在结直肠癌中的表达模式及其对细胞功能的影响。方法:采用qRT-PCR方法检测47例结直肠癌患者的癌组织和癌旁组织中miR-520e和星形胶质细胞上调基因-1(AEG-1)的mRNA表达水平。将SW480细胞分为对照组、miR-520e-mimic组、NC-mimic组、miR-520e-inhibitor组、NC-inhibitor组、miR-520e-mimic+AEG-1-pcDNA3.1组和miR-520e-mimic+NC-pcDNA3.1组。通过MTT法检测SW480细胞的增殖,通过Annexin V-FITC/PI双染色试剂盒检测细胞凋亡,通过Transwell检测细胞迁移和侵袭,通过双荧光素酶报告基因实验验证miR-520e和AEG-1的靶向关系,通过qRT-PCR或Western blotting检测AEG-1、基质金属蛋白酶2(MMP2)、MMP9、NF-κB p65(p65)和磷酸化的NF-κB p65(p-p65)的表达。结果:与癌旁组织相比,结直肠癌组织中miR-520e的表达水平降低(t=9.353,P<0.001)。与对照组相比,miR-520e-mimic组的OD值降低,细胞凋亡率升高,细胞迁移和侵袭数量降低,MMP2、MMP9和p-p65蛋白表达水平降低(P<0.001)。与对照组相比,miR-520e-inhibitor组的OD值升高,细胞凋亡率降低,细胞迁移和侵袭数量升高,MMP2、MMP9和p-p65蛋白表达水平升高。与NC-mimic组相比,miR-520e-inhibitor组的相对荧光素酶活性降低(P<0.001)。与对照组相比,miR-520e-mimic组的AEG-1的mRNA和蛋白表达水平均降低,而miR-520e-inhibitor组均升高(P<0.001)。与miR-520e-mimic+NC-pcDNA3.1组相比,miR-520e-mimic+AEG-1-pcDNA3.1组的AEG-1的mRNA和蛋白表达水平升高,OD值升高,细胞凋亡率降低,迁移和侵袭细胞数增加,MMP2和MMP9的蛋白表达水平及p65的磷酸化水平均增加(P<0.001)。结论:miR-520e在结直肠癌中表达降低,可通过靶向抑制AEG-1来发挥抗结直肠癌特性,其抗癌机制可能通过NF-κB信号通路介导。Objective: To reveal the expression pattern of MicroRNA-520e(miR-520e) in colorectal cancer and its effect on cell function. Methods: The qRT-PCR method was used to detect the expression levels of miR-520e and astrocyte elevated gene-1(AEG-1)mRNA in cancer tissues and adjacent tissues of 47 patients with colorectal cancer. SW480 cells were divided into the following groups:Control group, miR-520e-mimic group, negative control mimic group(NC-mimic group), miR-520e-inhibitor group, negative control inhibitor group(NC-inhibitor group), miR-520e-mimic+AEG-1-pcDNA3.1 group and miR-520e-mimic+NC-pcDNA3.1 group. SW480cells were transfected with Lipofectamine 2000 reagent. 48 h after transfection, the proliferation of SW480 cells was detected by MTT method, apoptosis was detected by Annexin V-FITC/PI double staining kit, cell migration and invasion were detected by Transwell, the target relationship between miR-520e and AEG-1 was verified by dual luciferase reporter gene experiment, and the expression of AEG-1,matrix metalloproteinase 2(MMP2), MMP9, NF-κB p65(p65) and phosphorylated NF-κB p65(p-p65) was detected by qRT-PCR or Western blotting. Results: Compared with adjacent tissues, the expression level of miR-520e in colorectal cancer tissues was reduced(t=9.353, P<0.001). Compared with the control group, the ODvalue of the miR-520e-mimic group decreased, the rate of cell apoptosis increased, the number of cell migration and invasion decreased, the expression level of MMP2, MMP9 and p-p65 protein decreased(P<0.001). Compared with the control group, the ODvalue of the miR-520e-inhibitor group increased, the apoptosis rate decreased, the number of cell migration and invasion increased, the expression level of MMP2, MMP9 and p-p65 protein increased(P<0.001). The dual luciferase reporter gene experiment showed that after co-transfection with WT-AEG-1-3’-UTR, the relative luciferase activity of the miR-520e-inhibitor group was reduced which compared with NC-mimic(P<0.001). Compared with the control group, the levels of

关 键 词：结直肠癌 MicroRNA-520e 星形胶质细胞上调基因-1 侵袭 NF-ΚB信号通路 

分 类 号：R-33[医药卫生] R735.3