Wei Zhong1, Jinchuan Yan2, Zhongqun Wang1, Yi Liang1. 1. Department of Cardiology, Affiliated Hospital of Jiangsu University, Zhenjiang 212001, China. 2. Department of Cardiology, Affiliated Hospital of Jiangsu University, Zhenjiang 212001, China; Email: yanjinchuan@hotmail.com.
Abstract
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-5p 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'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.
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-5p 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'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.