MiR-144通过靶向抑制GRK5表达促进脊髓星形胶质细胞活化

MiR-144 Promotes Activation of Astrocytes in Spinal Cord Injury by Targeting GRK5

尽管miR-144与细胞活化、增殖有关,但其对脊髓星形胶质细胞的作用尚不明确。本文旨在探究正常和脊髓损伤(spinal cord injury,SCI)组织和细胞中miR-144表达,以及miR-144能否通过靶向调节G蛋白偶联受体激酶5(GRK5)上调脊髓星形胶质细胞活化。实时定量PCR(RT-q PCR)和Western印迹结果揭示,与正常的组织/细胞相比,miR-144在脊髓损伤组织和星形胶质细胞中的表达水平显著降低,而GRK5的表达升高;脊髓损伤大鼠的星形胶质细胞中胶质原纤维酸性蛋白(GFAP)的表达显著低于正常大鼠,而GRK5蛋白的表达高于正常大鼠;MTT分析结果显示,转染miR-144可显著提高脊髓损伤大鼠的星形胶质细胞活性,但对细胞增殖无明显作用;酶活性分析发现,miR-144显著提高SOD和GSH活性;萤光素酶报告基因检测结果证明,miR-144能靶向结合GRK5,下调GRK5表达。miR-144 mimic转染或miR-144 mimic与pc DNA-GRK5共转染脊髓损伤的星形胶质细胞后,我们发现,miR-144转染可通过激活NF-κB通路消除GRK5对细胞活化的抑制作用。综上所述,miR-144通过靶向抑制GRK5促进脊髓星形胶质细胞的活化。

Although miR-144 is involved in the cell activation and proliferation, its role in spinal cord astrocytes remains unclear. In this study, we investigated the miR-144 expression in spinal cord astrocytes and tissues from spinal cord injury （SCI）and normal respectively, as well as whether miR-144 could promote the astrocyte activation by targeting G-protein-coupled receptor kinase 5 （GRK5）. Real- time q-PCR and Western blotting showed that miR-144 was decreased significantly in the spinal cord tissues and astroeytes of spinal cord injured rats, whereas GRK5 was increased inversely. Compared with the control, glial fibrillary acid protein （GFAP） expression was decreased while GRK5 was increased in the astrocytes derived from SCI rats. MTT assay revealed that miR-144 mimic transfection promoted the activation of astrocytes, but did not influence their proliferation. Meanwhile, the activities of SOD and GSH were enhanced by miR-144 mimic transfection as well. Bioinformaties analysis and biological luciferase reporter gene assay indicated that miR-144 targeted GRK5 and down-regulated the level of GRK5. When astroeytes were transfected with miR-144 mimic alone or co-transfected with miR-144 mimic and pcDNA-GRK5, we found that miR-144 rescued the inhibition of astroeytes activation induced by GRK5 via NF-KB activation. Collectively, miR-144 promoted activation of astroeytes in injured spinal cord by targeting GRK5.