microRNA-124-2在CD137-CD137L信号通路调控小鼠血管平滑肌细胞活化T细胞核因子C1表达中的作用

The Function of micro RNA-124-2 on CD137-CD137L Interaction Regulating Nuclear Factor of Activated T Cells C1 in Mouse Vascular Smooth Muscle Cells

目的探讨micro RNA-124-2在CD137-CD137L信号通路调控小鼠血管平滑肌细胞(VSMC)活化T细胞核因子C1(NFATc1)表达中的作用。方法小鼠血管平滑肌细胞采用组织贴块法原代培养,细胞中micro RNA-124-2表达采用RT-PCR法检测;应用脂质体转染技术将micro RNA-124-2的模拟物(mimic)和抑制物(inhibitor)转染至小鼠VSMC内,细胞中NFATc1 m RNA及蛋白表达采用实时荧光定量PCR及Western blot方法检测;应用双荧光素酶报告检测micro RNA-124-2对NFATc1-3'UTR的作用。结果 anti-CD137特异性刺激CD137-CD137L轴后,平滑肌细胞micro RNA-124-2表达较对照组降低(0.29±0.13比1.00±0.00,P<0.05),而anti-CD137L特异性阻断CD137-CD137L轴后,细胞中micro RNA-124-2表达较对照组增加(3.42±0.17比1.00±0.00,P<0.05);与阴性对照组相比,升高或下调micro RNA-124-2的表达均可逆转CD137-CD137L轴对NFATc1的作用;双荧光素酶报告系统显示micro RNA-124-2对NFATc1-3'UTR有抑制作用(0.283±0.011比1.294±0.143,P<0.001)。结论 CD137-CD137L受体-配体轴可以通过调控micro RNA-124-2进而影响NFATc1的表达。

Aim To study the function of microRNA-124-2 in the process of which CDI37-CD137L interaction regulates the NFATcl in mouse vascular smooth muscle cells. Methods With the stimulation/inhibition of CD137- CD137L axis by anti-CD137/anti-CD137L, RT-PCR technique was used to detect the expression of microRNA-124-2 in mouse vessel smooth muscle cells（VSMC）. Liposomal transfection was used to transfect microRNA-124-2 mimic and inhibitor into mouse VSMC, then the change of NFATcl mRNA and protein expressions was analyzed by qRT-PCR and Western blot after stimulated with anti-CD137/anti-CD137L in the corresponding group. Lueiferase Reporter Assay system was used to validate the role of microRNA-124-2 for NFATcl-3＇UTR. Results Compared with the control group,the expression of microRNA-124-2 had reduced after stimulated by anti-CD137 （ 0. 29 ± 0. 13 vs 1.00 ± 0. 00, P 〈 0. 05 ） , and increased by anti-CD137L（3.42 ±0. 17 vs 1.00±0. 00, P 〈0. 05 ）. The regulation of CD137-CDI37L axis to NFATcl could be reversed by transfected with mimic or inhibitor. Dual-Luciferase activity assay showed that microRNA-124-2 had an inhibition on NFATcl-3＇UTR（0.283 ±0.011 vs 1.294 ±0. 143,P 〈0.001）. Conclusions microRNA-124-2 could be regulated by CD137-CD137L interaction and thus affect NFATcl ＇ s expression.