Long noncoding RNA MIAT regulates VSMC migration by sponging miR-326

Abstract

ObjectiveThe current study aimed to investigate the role of the myocardial infarction-associated transcript (MIAT)/microRNA-326 (miR-326) axis in regulating the migration of vascular smooth muscle cells (VSMCs) during the progression of atherosclerosis (AS).MethodsBioinformatic analysis of MIAT and miR-326 in two AS-related GEO datasets was performed via the online web tool GEO2R. MIAT and miR-326 expression in 46 paired plasma samples and in oxidized low-density lipoprotein (ox-LDL)-treated VSMCs was analysed via RT-qPCR. Western blot analysis was used to determine the expression of monocyte chemotactic protein 1 (MCP-1) after diverse ox-LDL treatments. The correlation between MIAT and miR-326 was analysed by Spearman correlation analysis. Transwell assays were performed to determine the changes in migration after different MIAT or miR-326 interventions. RNA-fluorescence in situ hybridization (FISH) assays were performed to determine the subcellular localization of MIAT and miR-326. The targeted binding effect between MIAT and miR-326 was confirmed via a luciferase assay.ResultsMIAT was upregulated and miR-326 was downregulated in 46 plasma samples from patients with AS compared with those from patients without AS (non-AS). A negative correlation was found between MIAT and miR-326 (r = - 0.6591, P < 0.0001). The expression of MIAT in plaque samples from advanced AS patients was markedly greater than that in plaque samples from early AS patients according to the GEO dataset GSE28829 (P < 0.0001). The expression of miR-326 in platelet samples from patients with first acute myocardial infarction (FAMI) was significantly lower than that in healthy controls (P = 0.0034). MCP-1 was upregulated in ox-LDL-treated VSMCs. MIAT knockdown by specific MIAT small interfering RNAs (siRNAs) suppressed VSMC migration. Upregulation of miR-326 by transfection of miR-326 mimics also inhibited VSMC migration. Dual luciferase assays indicated that miR-326 targets MIAT. The upregulation of MIAT increased the migration of VSMCs. However, this effect was attenuated by a miR-326 mimic.ConclusionsMIAT was upregulated and miR-326 was downregulated in AS plasma and in ox-LDL-treated VSMCs. MIAT binds to miR-326 via theoretical miRNA response elements. MIAT promoted migration by sponging miR-326 in ox-LDL-induced VMSCs. The MIAT/miR-326 axis may represent a novel therapeutic target for the treatment of AS, offering potential insights into AS progression and its clinical management.

Keywords: AS; MIAT; VSMCs; ceRNA; miR-326; migration.

Figure 1.

MIAT was upregulated in patients with AS and ox-LDL-treated VSMCs. (a) The DEGs associated with advanced AS plaques and early AS plaques in the AS-related GEO dataset GSE28829 were analysed using GEO2R. (b) The expression of MIAT in 16 advanced AS plaque samples and in 13 early AS plaque samples. Probe ID 228658_at represents MIAT. (c) The expression of MIAT in 16 advanced AS plague samples was significantly greater than that in 13 early AS plague samples. ****P < 0.0001. (d) MIAT expression was upregulated in most (41/46, 89.13%) plasma samples from patients with AS, as detected via RT–qPCR. ^****P < 0.0001. (e) MIAT expression in 46 plasma samples from patients with AS and in 46 paired plasma samples from patients without AS, as determined via RT–qPCR. ^****P < 0.0001. (f) MCP-1 expression in ox-LDL-treated VSMC models. ****P < 0.0001. Con, treated with double distilled water; low-AS, treated with 10 μg/mL ox-LDL; high-AS, treated with 50 μg/mL ox-LDL. (g) The expression of MIAT in ox-LDL-treated VSMC models was measured via RT–qPCR. ****P < 0.0001.

Figure 2.

MIAT knockdown suppressed the migration of ox-LDL-treated VSMCs. (a) The expression of MIAT in ox-LDL-treated VSMC models after transfection with scramble small interfering RNAs (scramble siRNA, siSCR) or specific siRNAs targeting MIAT (siMIAT-1 and siMIAT-2) was confirmed via RT–qPCR. ^**P < 0.01. (b) Cell migration changes after the transfection of ox-LDL-treated VSMC models with siRNAs targeting MIAT. **P < 0.01. Magnification:×400; scale bar: 200 μm.

Figure 3.

miR-326 was downregulated in AS- and ox-LDL-treated VSMCs. (a) The differentially expressed miRNAs in 17 platelet samples from patients with FAMI and in 17 matched platelet samples from control patients were analysed in GSE24548 via GEO2R with the filtration criterion of adjusted P(adj) < 0.05. (b) The predicted miRNAs that might interact with MIAT in the LncBase and miRDB databases overlapped with the identified miRNAs in GSE24548. miR-326 was selected. (c-d) The expression of miR-326 in platelet samples of FAMI and in platelet samples of the control is presented. **P < 0.01. (e) The differential expression of miR-326 in 46 plasma samples from patients with AS and in 46 paired plasma samples from patients without AS was quantified via RT–qPCR. ****P < 0.0001. (f) The relationship between MIAT and miR-326 in 46 plasma samples from patients with AS was analysed via Spearman correlation analysis. r = − 0.6591, P < 0.0001. (g) The expression of miR-326 in ox-LDL-treated VSMC models was determined via RT–qPCR. ^****P < 0.0001, *P < 0.05. (h) The expression of miR-326 after transfection of miR-326 mimics and corresponding negative control mimics (NC mimics) in ox-LDL-treated VSMC models was determined via RT–qPCR. ^****P < 0.0001. (i) Cell migration changes after diverse miR-326 interventions in ox-LDL-treated VSMC models were evaluated by a Transwell assay. Magnification:×400; scale bar: 200 μm. **P < 0.01.

Figure 4.

MIAT sponged miR-326 to facilitate the migration of ox-LDL-treated VSMCs. (a) MIAT and miR-326 were colocalized in the cytoplasm of VSMCs, as determined by a FISH assay. (b) MIAT supplies theoretical miR-326 response elements (MRE-326) as predicted by DIANA-lncBase. (c) Diagram of the wild-type and mutated luciferase reporter plasmids. (d) The targeted binding effect between MIAT and miR-326 was verified by a luciferase assay. **P < 0.01, ^n.sP > 0.05. (e) Cell migration changes after diverse MIAT and miR-326 interventions in ox-LDL-treated VSMC models were evaluated via a Transwell assay. Magnification:×400; scale bar: 200 μm. ****P < 0.0001, ^n.sP > 0.05.