NEDD4L增强IGF2BP2的泛素化对结直肠癌细胞SW620增殖、凋亡、迁移和侵袭的影响

Effects of NEDD4L enhancing IGF2BP2 ubiquitination on proliferation,apoptosis,migration and invasion of colorectal cancer cells SW620

目的:探讨NEDD4L对人转移性结直肠癌细胞SW620迁移和侵袭的影响及其可能的作用机制。方法:生物信息学分析筛选IGF2BP2/NEDD4L泛素化作用轴并构建NEDD4L过表达的SW620细胞株,实时荧光定量(RT-qPCR)检测NEDD4L mRNA表达水平;CCK-8法和流式细胞术检测细胞增殖和凋亡;细胞划痕和Transwell实验检测细胞迁移和侵袭,CHX和MG132处理SW620细胞,Western blot检测IGF2BP2蛋白的表达。同时构建Flag-IGF2BP2、His-NEDD4L、HA-Ub重组质粒并在293T细胞中采用免疫共沉淀检测NEDD4L对IGF2BP2的泛素化水平的影响。结果:SW620细胞中成功实现NEDD4L过表达,并且导致细胞增殖能力显著降低(P<0.05),凋亡能力显著上升(P<0.01);同时抑制细胞周期进程(P<0.05)。细胞迁移率显著降低(P<0.01),细胞侵袭数量显著降低(P<0.01);Western blot结果显示,过表达NEDD4L能够显著降低IGF2BP2、YAP、VEGFA的蛋白表达水平,Co-IP结果显示NEDD4L增强IGF2BP2与Ub的结合能力。结论:NEDD4L在结直肠癌细胞中通过增强IGF2BP2的泛素化,进而降低YAP、VEGFA的蛋白表达来实现抑癌功能。

Objective:To investigate the effect of NEDD4L on the migration and invasion of human metastatic colorectal cancer cell SW620 and its possible mechanism.Methods:Bioinformatics analysis was used to screen the IGF2BP2/NEDD4L ubiquitination axis and construct SW620 cell line with NEDD4L overexpression.NEDD4L mRNA expression level was detected by real-time fluorescence quantitative PCR(RT-qPCR).Cell proliferation and apoptosis were detected by CCK-8 method and flow cytometry.Cell scratch and Transwell assays were used to detect cell migration and invasion.SW620 cells were treated with CHX and MG132,and the expression of IGF2BP2 protein was detected by Western blot.The recombinant plasmids of Flag-IGF2BP2,His-NEDD4L and HA-Ub were constructed,and the effect of NEDD4L on the ubiquitination level of IGF2BP2 was detected by co-immunoprecipitation in 293T cells.Results:NEDD4L overexpression was successfully achieved in SW620 cells,and the cell proliferation ability was significantly decreased(P<0.05),and the apoptosis ability was significantly increased(P<0.01),simultaneous inhibition of cell cycle progression(P<0.05).The cell migration rate and the number of cell invasion were significantly decreased(P<0.01).Western blot results showed that overexpression of NEDD4L significantly reduced the protein expression levels of IGF2BP2,YAP and VEGFA.Co-IP results showed that NEDD4L enhanced the binding ability of IGF2BP2 to Ub.Conclusion:NEDD4L function as a tumor suppressor in colorectal cancer cells by enhancing the ubiquitination of IGF2BP2 and reducing the protein expressions of YAP and VEGFA.