激活大麻素受体1诱导的单核巨噬细胞J774A.1的迁移依赖RNA结合蛋白HuR

Mouse monocyte / macrophage cell line J774A.1 migration induced by the activation of CB1 depends on HuR

目的研究大麻素受体1(cannabinoid receptor 1,CB1)在单核巨噬细胞迁移中的重要作用以及RNA结合蛋白人抗原R(human antigen R,HuR)参与其中的可能机制。方法选用单核巨噬细胞系J774A.1,应用琼脂糖凝胶电泳和免疫荧光染色技术鉴定J774A.1中CB1以及HuR的表达;ACEA和AM281分别为CB1的药理学激动剂和拮抗剂,应用Boyden chamber法检测ACEA和AM281对J774A.1迁移活性的影响。HuR的基因干扰用于确定激活CB1诱导的J774A.1迁移功能是否依赖HuR;胞质蛋白的分离用于探究激活CB1是否能引起J774A.1胞质中HuR的富集;RT-qPCR和Western blotting法检测CB1和HuR mRNA和蛋白质的变化情况。结果该研究证明J774A.1在基因和蛋白质水平上均表达CB1和HuR;激活CB1能够促进J774A.1的迁移(P<0.01)并且能够被其药理学拮抗剂AM281所抑制;激活CB1诱导的J774A.1的迁移依赖HuR;激活CB1促进了J774A.1胞质中HuR的富集进一步影响了CB1的表达,由此HuR参与了激活CB1诱导的J774A.1的迁移。结论激活CB1能够诱导单核巨噬细胞系J774A.1的迁移,且此过程依赖RNA结合蛋白HuR。

Objective To investigate the effect of activation of CB1 on J774A. 1 migration and to know whether HuR is involved in the process. Methods Immunofluorescence and agarose gel electrophoresis were used to detect the expression of cannabinoid receptor 1 （ CB1 ） and human antigen R （HuR）. Boyden chamber was used for cell migration assay. HuR siRNA transfection was used to determine whether ACEA（ CB1 agonist） induced-migration depended on HuR. In order to observe its underlying mechanisms, HuR subcellular localization was assayed by Western blotting of cellular fractions from J774A. 1 stimulated with ACEA. The effects of HuR siRNA on CB1 mRNA and protein levels in J774A. 1 stimulated with ACEA was assayed by Western blotting. Results In this study, we present evidence that J774A. 1 significantly expressed HuR and CB1 ; the treatment of ACEA caused a increase in J774A. 1 migration （P 〈 0. 01 ）, and pharmacological inhibition of CB1 markedly attenuated ACEA-induced migration; ACEA-induced J774A. 1 migration depended on HuR. Furthermore, we demonstrated ACEA increased HuR cytoplasmic translocation, and HuR regulated the expression of CB1. Conclusion The activation of CB1 induces J774A. 1 migration and HuR is involved in the process.