MicroRNA-124-3p inhibits collagen synthesis in atherosclerotic plaques by targeting prolyl 4-hydroxylase subunit alpha-1 (P4HA1) in vascular smooth muscle cells

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.