CD137分子通过微小RNA-145a-5p调控载脂蛋白E-/-小鼠活化T细胞核因子c1表达

CD137 signaling regulates the expression of nuclear factor of activated T cells c1 through miR-145a-5p in ApoE-/-mice

目的 探讨CD137分子信号通路是否通过微小RNA-145a-5p （miR-145a-5p）调控活化T细胞核因子c1（NFATc1）表达.方法 选取18只载脂蛋白（Apo）E-/-小鼠,8周龄,颈动脉硅胶圈植入建立模型后,根据随机区组法分为对照组、CD137刺激组和CD137抑制组,每组6只;采用荧光定量PCR（qRT-PCR）检测小鼠颈动脉斑块以及平滑肌细胞中miR-145a-5p含量;免疫荧光检测斑块中NFATc1分布,qRT-PCR、Western blot方法检测小鼠体内、外NFATc1表达.用脂质体转染miR-145a-5p的拟似物和抑制物进入血管平滑肌细胞（VSMC）;通过分子克隆技术构建NFATc1真核表达载体p3xFLAG-NFATc1、p3xFLAG-NFATc 1-3＇ UTR.构建双荧光素酶报告载体psicheck2-NFATc1,并通过同源重组法构建psicheck2-NFATc1的突变载体psicheck2-NFATc1-Mut转染细胞后分为正常组和突变组.结果 CD137刺激组ApoE-/-小鼠斑块及VSMC中miR-145a-5p水平明显低于对照组（分别为0.21 ±0.06比1.00±0.00,P＜0.05;0.22±0.07比0.50±0.12,P＜0.05）,斑块内NFATc1表达高于对照组（P ＜0.05）;CD137抑制组ApoE-/-小鼠斑块及VSMC中miR-145a-5p水平低于CD137刺激组（分别为0.74±0.17比0.21 ±0.06,P＜0.05;0.78 ±0.12比0.22 ±0.07,P＜0.05）,而斑块内NFATc1表达高于CD137刺激组（P＜0.05）.miR-145a-5p的拟似物处理组中NFATc1表达低于对照组,而抑制物处理组中NFATc1表达高于对照组（P＜0.05）;p3xFLAG-NFATc1-3＆#39; UTR过表达组中外源性NFATc1表达低于对照组（P＜0.05）;双荧光素酶实验中,正常组中拟似物处理组荧光水平明显低于对照组（0.56±0.08比1.00 ±0.00,P＜0.05）,突变组中荧光水平与对照组比较差异无统计学意义.结论 CD137分子能够通过miR-145a-5p调控NFATc1的表达.

Objective To investigate if miR-145a-5p participates the modulation process of CD137 signaling on the expression of nuclear factor of activated T cells c1 （NFATc1） in ApoE-/-mice.Methods Atherosclerotic plaque model was produced by perivascular carotid collar placement in ApoE-/-mice.After surgery, the mice were randomly divided into the following groups： CD137 activated group （CD137 group, n =6） ,CD137 inhibited group （anti-CD137 group, n =6） and control group（n =6）.The mRNA expression of miR-145a-Sp in plaque and cells was measured by real-time quantitative PCR （RT-PCR）.Immunofluorescence was used to observe the distribution of NFATc1 in plaque and the expression of NFATc1 at mRNA and protein levels were detected by qRT-PCR, Western blot, respectively.The mouse vascular smooth muscle cells （VSMCs） were isolated and transfected with miR-145a-5p mimics or inhibitors by Lipofectamine.The eukaryotic expression vector and luciferase vector including p3xFLAG-NFATc1, p3xFLAG-NFATc1-3＆#39;UTR,psicheck2-NFATc1, psicheck2-NFATc1-Mut were constructed through molecular cloning and homologous recombination techniques, 293T cells were transfected with the miR-145a-5p mimics or inhibitors and the protein level and fluorescence intensity were then measured, respectively.Results In vivo or in vitro, the level of miR-145a-5p was significantly decreased （0.21 ± 0.06 vs.1.00 ± 0.00, P 〈0.05,0.22 ± 0.07 vs.0.50 ± 0.12, P 〈 0.05） while the opposite effects were observed in anti-CD137 group.NFATc1 expression was decreased or increased in VSMCs transfected with miR-145a-5p mimics or inhibitors, respectively （all P 〈 0.05）.miR-145a-5p mimics decreased the expression of p3xFLAG-NFATc1-3＇UTR and the fluorescence intensity （0.56 ± 0.08 vs.1.00 ± 0.00, P 〈 0.05）.Conclusion CD137 signaling participates the regulation process on the expression of NFATc1 through miR-145a-5p in ApoE-/-mice.