miR-126-5p靶向Notch2 对结肠癌SW480 细胞增殖、迁移、侵袭和凋亡的影响

目的:探讨miR-126-5p 对结肠癌SW480 细胞增殖、迁移、侵袭和凋亡的影响及其作用机制。方法：用miR-126 mimic和pcDNA Notch2（pc-Notch2）等分别或同时转染结肠癌SW480 细胞，用qPCR法检测miR-126-5p 和Notch2 的表达；荧光素酶报告实验观察miR-126-5p 和Notch2 的靶向关系；CCK-8 法、划痕愈合实验、Transwell 小室法和Annexin V/PI 染色流式细胞术分别检测转染细胞的增殖、迁移、侵袭和凋亡，Western blotting 检测Notch2、增殖细胞核抗原（proliferating cell nuclear antigen, PCNA）、cleaved Caspase-3、MMP-2 和MMP-9 的表达。结果：转染miR-126 mimic 能显著升高SW480 细胞miR-126-5p 的表达水平（P<0.01）和显著抑制SW480 细胞Notch2 的表达（P<0.01），同时证实Notch2 上存在miR-126-5p 的结合位点。上调miR-126-5p 显著抑制SW480 细胞增殖并降低PCNA的表达水平（P<0.01）、升高细胞凋亡率和cleaved Caspase-9 的表达水平（均P<0.01），pc-Notch2显著减弱miR-126 mimic 对SW480 细胞增殖和凋亡的调控作用；miR-126 mimic 显著降低SW480 细胞划痕愈合率和穿膜细胞数（均P<0.01）、抑制MMP-2 和MMP-9 的表达（P<0.01）；pc-Notch2 显著减弱miR-126 mimic 对SW480 细胞迁移、侵袭及MMP-2、MMP-9表达的抑制作用（均P<0.01）。结论：miR-126-5p 通过抑制Notch2 表达，降低结肠癌SW480 细胞增殖、迁移和侵袭能力。

Effects of miR-126-5p on proliferation, migration, invasion and apoptosis of colon cancer SW480 cells via targeting Notch2

Objective: To investigate the effects and mechanisms of miR-126-5p on proliferation, migration, invasion and apoptosis of colon cancer SW480 cells. Methods: Cells were transferred with miR-126 mimic and pcDNA Notch2 (pc-Notch2) respectively or simultaneously.Real-time fluorescence quantitative PCR was performed to detect the expression of miR-126 and Notch2. The relationship of miR-126-5p and Notch2 was determined by luciferase reporter assay. The CCK-8 assay, wound healing assay, Transwell and flow cytometry were performed to examine cell proliferation, migration, invasion and apoptosis, respectively. The protein levels of Notch2, proliferating cell nuclear antigen (PCNA), cleaved Caspase-3, metalloproteinase-2 (MMP-2) and MMP-9 were measured by Western blotting. Results: miR-126 mimic significantly increased expression level of miR-126-5p but reduced the expression of Notch2 in SW480 cells (all P<0.01); in the meanwhile, a binding site with miR-126-5p was confirmed on Notch2. Up-regulating the expression of miR-126-5p inhibited cell proliferation and the expression of PCNA (P<0.01), increased the cell apoptosis rate and protein level of cleaved Caspase-3 notably (all P<0.01). Pc-Notch2 obviously alleviated the effects of miR-126 mimic on cell proliferation and apoptosis (all P<0.01). Furthermore, miR-126 mimic significantly decreased the wound healing rate and invasive cell numbers (all P<0.01),and down-regulated the expressions of MMP-2 and MMP-9 (P<0.01); pc-Notch2 alleviated the effects of miR-126 mimic on cell migration,invasion and the expressions of MMP-2 and MMP-9 (all P<0.01). Conclusion: miR-126-5p can attenuate proliferation, migration and invasive ability of colon SW480 cells via inhibiting the expression of Notch2.