In silico and in vitro analysis of microRNAs with therapeutic potential in atherosclerosis

Abstract

Atherosclerosis is a chronic inflammatory disease in which aberrant lipid metabolism plays a key role. MicroRNAs (miRNAs), micro-coordinators of gene expression, have been recently proposed as novel clinical biomarkers and potential therapeutic tools for a broad spectrum of diseases. This study aimed to identify miRNAs with therapeutic potential in atherosclerosis. Bioinformatic databases, including experimentally validated and computational prediction tools as well as a novel combination method, were used to identify miRNAs that are able to simultaneously inhibit key genes related to the pathogenesis of atherosclerosis. Further validation of genes and miRNAs was conducted using the STRING online tool, KEGG pathway analysis and DIANA-miRPath. The inhibitory effects of the identified miRNAs in HepG2 and Huh7 cells were verified by real-time PCR. The MTT assay was utilized to evaluate cell cytotoxicity effects of miRNAs. Atherosclerotic drug-targeted genes were selected as key genes. Strong interactions between genes were confirmed using STRING. These genes were shown to be integral to critical pathological processes involved in atherosclerosis. A novel combined method of validated and predicted tools for the identification of effective miRNAs was defined as the combination score (C-Score). Bioinformatic analysis showed that hsa-miR-124-3p and hsa-miR-16-5p possessed the best C-Score (0.68 and 0.62, respectively). KEGG and DIANA-miRPath analysis showed that selected genes and identified miRNAs were involved in atherosclerosis-related pathways. Compared with the controls in both HepG2 and Huh7 cell lines, miR-124 significantly reduced the expression of CETP, PCSK9, MTTP, and APOB, and miR-16 significantly reduced the expression of APOCIII, CETP, HMGCR, PCSK9, MTTP, and APOB, respectively. The cytotoxicity assay showed that miR-124 reduced cell viability, especially after 72 h; however, miR-16 did not show any significant cytotoxicity in either cell line. Our findings indicate that hsa-miR-124 and miR-16 have potential for use as therapeutic candidates in the treatment of atherosclerosis.

MiR-16 is promising to be a therapy target to alleviate atherosclerosis

“…Some recent studies have directly linked miR-16 with the pathogenesis of atherosclerosis. Mahjoubin-Tehran M, et al reports that, miR-16 can modulate apolipoprotein B (APOB), proprotein convertase subtilisin/kexin type 9 (PCSK9), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), all of which have been confirmed as therapy targets of atherosclerosis by clinical trials; importantly, this study preliminarily reveals that miR-16 represses interleukin-1 beta (IL-1β, another important player in inflammation during atherosclerosis), and miR-16 shows low cytotoxicity for both tested hepatic cell lines [9]. Wang M, et al demonstrates that miR-16 was lowly expressed in the plasma and peripheral blood monocytes of patients with coronary artery disease, and in vivo experiments show that injection of miR-16 agomiR in ApoE-/-mice reduced the formation of atherosclerotic plaque and inhibits the release of proinflammatory factors including IL-6, TNF-α, MCP-1, and IL-1β [10].…”

Identification of novel prognostic targets in coronary artery disease and related complications using bioinformatics and next generation sequencing data analysis

“…Previous study showed that DDX3X [348], EGR1 [350] and SREBF1 [539] were associated with non-alcoholic fatty liver disease. EGR1 [84], HIF1A [88], STAT1 [60], hsa-mir-16-5p [540], hsa-mir-532-3p [541], hsa-mir-503-5p [542], SREBF1 [543] and FOXP3 [544] plays a pivotal role in the atherosclerosis. The results showed that EGR1 [119] expression participate in the progression of cardiac arrhythmia.…”

New Insight into Mechanisms of Cardiovascular Diseases: An Integrative Analysis Approach to Identify TheranoMiRNAs

“…The use of in silico tools may be considered fundamental in this research field [ 16 , 17 , 18 ]. A recent article demonstrated the involvement of hsa-miR-124 and miR-16 in the biomolecular mechanisms of atherosclerosis, suggesting their potential use as diagnostic and therapeutic biomarkers (theranoMiRNAs) [ 19 ]. Another experimental study, using an integrated approach with an in silico analysis, demonstrated the involvement of different miRNAs in coronary artery disease (CAD) [ 20 ].…”

“…In this context, it seems clear that the molecular mechanisms underlying miRNA functions are not completely clear. Moreover, to date, in silico studies could be very useful to clarify several molecular aspects, suggesting future directions for in vivo studies [ 8 , 19 , 21 , 22 , 23 ]. This study aims to explore different bioinformatic tools in order to clarify miRNA interactions with candidate genes, showing the need to use a computational approach in establishing the most probable associations between miRNAs and target genes.…”