基于FAK/ERK途径探讨NRP-1对结直肠癌细胞的影响

Effects of NRP-1 on Colorectal Cancer Cells Based on FAK/ERK Pathway

目的探究神经纤毛蛋白-1(neuropilin-1,NRP-1)对结直肠癌(colorectal cancer,CRC)细胞行为的作用,及其对黏着斑激酶(focal adhesion kinase,FAK)/胞外信号调节激酶(extracellular signal-regulated kinases,ERK)信号途径的影响。方法将CRC细胞系DLD-1随机分为对照组、CT-707组、sh-NRP-1组、sh-NRP-1+CT-707组,按分组使用sh-NRP-1慢病毒液感染细胞与FAK抑制剂CT-707处理细胞,通过荧光显微镜观察感染慢病毒后细胞内增强型绿色荧光蛋白表达情况,实时荧光定量PCR和Western blot法检测细胞内NRP-1mRNA与蛋白的表达变化。CCK-8法和EdU染色检测细胞增殖能力,细胞划痕实验观察细胞划痕愈合率,Transwell实验检测细胞迁移与侵袭,Western blot法测定细胞内FAK/ERK相关蛋白表达情况。结果感染sh-NRP-1重组慢病毒的DLD-1细胞中可见明显绿色荧光,细胞内NRP-1mRNA相对表达量和蛋白相对表达量均显著下调(P<0.05),表明转染成功。与对照组比较,CT-707组和sh-NRP-1组的细胞增殖活性显著下降,EdU阳性百分比显著降低,细胞划痕愈合率显著降低,细胞迁移数目与侵袭数目均显著减少,FAK蛋白磷酸化水平和ERK蛋白磷酸化水平均显著下调(P<0.05);与sh-NRP-1组比较,sh-NRP-1+CT-707组细胞增殖活性显著下降,EdU阳性百分比和细胞划痕愈合率均显著降低,细胞迁移数目与侵袭数目也均显著减少,FAK蛋白磷酸化水平和ERK蛋白磷酸化水平均显著下调(P<0.05)。结论下调NRP-1表达能够发挥抑制CRC细胞增殖、迁移及侵袭的作用,该机制可能与调控FAK/ERK信号转导途径有关。

Objective To investigate the effect of neuropilin-1(NRP-1)on the metastasis of colorectal cancer(CRC)cells,and its effect on focal adhesion kinase(FAK)/extracellular signal-regulated kinases(ERK)signaling pathway.Methods The CRC cell lines DLD-1 were randomly divided into control group,CT-707 group,sh-NRP-1 group,and sh-NRP-1+CT-707 group.The cells were infected with sh-NRP-1 lentiviral solution according to the groups.Cells were treated with the FAK inhibitor CT-707,the expression of enhanced green fluorescent protein in cells after infection with lentivirus was observed by fluorescence microscope.The expression changes of NRP-1mRNA and protein in cells were detected by real-time fluorescence quantitative PCR and western blot.The proliferation ability of cells was detected by CCK-8 method and EdU staining.The cell scratch experiment was applied to observe the healing rate of cell scratches,and Transwell assay was used to detect cell migration and invasion.Western blot was used to measure the protein expressions of FAK/ERK pathway.Results Significant green fluorescence was observed in DLD-1 cells infected with sh-NRP-1 recombinant lentivirus,and the relative expression of NRP-1mRNA and protein in the cells were significantly down-regulated(P<0.05),indicating that the transfection was successful.Compared with the control group,the cell proliferation activity,the percentage of EdU positive,the cell wound healing rate,and the number of cell migration and invasion in the CT-707 group and the sh-NRP-1 group were significantly decreased,and the phosphorylation levels of FAK protein and ERK protein were significantly down-regulated(P<0.05);compared with the sh-NRP-1 group,the cell proliferation activity,the percentage of EdU positive,the wound healing rate of the cells,and the number of cell migration and invasion in the sh-NRP-1+CT-707 group were significantly decreased,and the phosphorylation levels of FAK protein and ERK protein were significantly down-regulated(P<0.05).Conclusion Down-regulation of NRP-1 expression can inhibit the proliferation,migration and invasion of CRC cells.This mechanism may be related to the regulation of FAK/ERK signal transduction pathway.