Identification and Functional Validation of the miRNAs and their Targets, which are Essential to the Induction of Neointimal Hyperplasia

Abstract

Angioplasty and stent procedures for widening the narrowed artery of atherosclerosis can cause restenosis by damaging endothelial cells. The main reason for this phenomenon is the migration and proliferation of vascular smooth muscle cells toward the intimal layer, which is called neointimal hyperplasia. After the function of miRNA, which is cell‐specific and fine‐tunes the translation of several mRNA targets, was known, several studies have identified miRNAs that affect arterial restenosis to overcome the limits of the previous drugs. However, most of the studies focused on one miRNA each. Therefore, it is necessary to ensure which of the individually studied miRNAs play a more important role at the beginning of restenosis. We should also ensure that there are any unknown miRNAs associated with restenosis regulation. In this study, we used the rat balloon injured carotid artery model to identify the entire differentially expressed (DE)‐miRNAs and DE‐mRNAs in the early stages of restenosis, through small RNA sequencing and total RNA sequencing, respectively. Among the DE‐miRNAs which show the same results in small RNA sequencing and RT‐qPCR, we focus on the rat, human conserved miRNAs with the largest change in expression (> 5 fold). The conserved DE‐miRNAs included previously well‐known miRNAs, such as miR‐221 and miR‐21, but also miRNAs, which show more change in expression but still unknown. By performing in vivo and in vitro functional assays, we were able to identify several undiscovered miRNAs that are effective in the cellular functions associated with the suppression of neointimal hyperplasia. Next, among the DE‐mRNAs identified by total RNA sequencing, we selected the predicted targets of the miRNAs through the target prediction databases (Targetscan, miRDB, miRmap). Also, we selected mRNAs functionally in context with the miRNAs by using the Gene Ontology (GO) term of the expected targets. We used RT‐qPCR to double‐check the changes in expression of these filtered targets and to confirm whether these predicted targets are actual targets for miRNAs. For the mRNA with the greatest variation, we performed luciferase assay to check whether the mRNA is a direct target of the miRNA. Additional functional assays were used to determine whether the target mRNA plays a central role in the progression of the neointimal hyperplasia.In summary, we identified entire miRNAs and their target mRNAs that are important for restenosis control. Through this study, we expect to utilize diverse miRNAs for the diagnosis and therapy of restenosis.Support or Funding InformationThis study was supported by the grant from the National Research Foundation of Korea (2018029571)