Wei'jia Chen1, Fangpu Yu1, Mingxue Di1, Mengmeng Li1, Yifei Chen1, Yu Zhang1, Xiaolin Liu1, Xiaozhen Huang1, Mei Zhang2. 1. The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China. 2. The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China. Electronic address: daixh@vip.sina.com.
Abstract
BACKGROUND AND AIMS: Collagen synthesis in vascular smooth muscle cells (VSMCs) is very important in atherosclerosis, as it affects plaque stability. In this study, we aim to assess whether miR-124-3p is involved in the collagen synthesis process in VSMCs and the role it might play in atherosclerotic development. METHODS: We modulated the miR-124-3p expression in the aortic root plaques of high-fat-diet fed ApoE-/- mice by lentivirus injection. To determine plaque size and the content of plaque-stability-related cells or molecules, stainings, including hematoxylin and eosin, Oil red O, Sirius Red and immunohistochemical staining, were performed. Fluorescence in situ hybridization (FISH) was used to locate miR-124-3p in atherosclerotic plaques. Western blotting and RT-qPCR were carried out to determine the level of P4HA1 as well as type I and type III collagen protein and mRNA expression. RESULTS: Results showed that collagen and VSMC content of plaques was inversely correlated with miR-124-3p levels. By FISH, we identified that miR-124-3p was primarily expressed by VSMCs. We also found that protein levels of type I and type III collagen in aortas and atherosclerotic plaques were decreased by miR-124-3p. We modulated miR-124-3p level in vitro and found it could inhibit collagen expression in HASMCs. This might be caused by the downregulation of P4HA1. P4HA1 was predicted as miR-124-3p's direct target, which was verified with a dual luciferase reporter assay and RIP test. CONCLUSIONS: The results presented here provide evidence that miR-124-3p inhibits VSMC collagen synthesis by directly targeting P4HA1, which might decrease atherosclerotic plaque stability.
BACKGROUND AND AIMS: Collagen synthesis in vascular smooth muscle cells (VSMCs) is very important in atherosclerosis, as it affects plaque stability. In this study, we aim to assess whether miR-124-3p is involved in the collagen synthesis process in VSMCs and the role it might play in atherosclerotic development. METHODS: We modulated the miR-124-3p expression in the aortic root plaques of high-fat-diet fed ApoE-/- mice by lentivirus injection. To determine plaque size and the content of plaque-stability-related cells or molecules, stainings, including hematoxylin and eosin, Oil red O, Sirius Red and immunohistochemical staining, were performed. Fluorescence in situ hybridization (FISH) was used to locate miR-124-3p in atherosclerotic plaques. Western blotting and RT-qPCR were carried out to determine the level of P4HA1 as well as type I and type III collagen protein and mRNA expression. RESULTS: Results showed that collagen and VSMC content of plaques was inversely correlated with miR-124-3p levels. By FISH, we identified that miR-124-3p was primarily expressed by VSMCs. We also found that protein levels of type I and type III collagen in aortas and atherosclerotic plaques were decreased by miR-124-3p. We modulated miR-124-3p level in vitro and found it could inhibit collagen expression in HASMCs. This might be caused by the downregulation of P4HA1. P4HA1 was predicted as miR-124-3p's direct target, which was verified with a dual luciferase reporter assay and RIP test. CONCLUSIONS: The results presented here provide evidence that miR-124-3p inhibits VSMC collagen synthesis by directly targeting P4HA1, which might decrease atherosclerotic plaque stability.
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