| 吕飒美,张 健,吴友伟,刘 倩,易 默.MicroRNA-520e通过靶向抑制AEG-1发挥抗结直肠癌特性[J].,2022,(17):3229-3237 |
| 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 |
| 投稿时间：2021-03-03 修订日期：2021-03-26 |
| DOI：10.13241/j.cnki.pmb.2022.17.006 |
| 中文关键词: 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 |
| 英文关键词: Colorectal cancer MicroRNA-520e Astrocyte elevated gene-1 Invasion NF-κB signaling pathway |
| 基金项目:陕西省自然科学基础研究计划项目（2018JM7118） |
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| 中文摘要: |
| 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OD490nm 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OD490nm 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，OD490nm 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。 |
| 英文摘要: |
| ABSTRACT 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. SW480 cells 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 OD490nm value 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 OD490nm value 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 AEG-1 mRNA and protein expression in the miR-520e-mimic group were reduced, while those in the miR-520e-inhibitor group were increased (P<0.001). Compared with the miR-520e-mimic+NC-pcDNA3.1 group, the expression levels of AEG-1 mRNA and protein in miR-520e-mimic+AEG-1-pcDNA3.1 group increased, the OD490nm value increased, the apoptosis rate decreased, the number of migrating and invasive cells increased, the protein expression levels of MMP2 and MMP9, as well as the phosphorylation level of p65 increased (P<0.001). Conclusion: The expression of miR-520e is reduced in colorectal cancer, miR-520e exerts anti-colorectal cancer properties by targeting AEG-1, and its anti-cancer mechanism may be mediated by NF-κB signaling pathway. |
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