Dysregulation of the Urothelial Cancer Associated 1 Long Noncoding RNA Promotes Proliferation of Vascular Smooth Muscle Cells by Modulating Expression of P27KIP1/CDK2.

Background: Atherosclerosis is one of the leading causes of morbidity and mortality worldwide. A variety of long noncoding RNAs (lncRNAs) have been reported to be significantly involved in vascular smooth muscle cell (VSMC) proliferation, which is an essential process for atherosclerotic plaque formation. The aim of this study was to investigate the mechanism of lncRNA urothelial cancer associated 1 (UCA1) involvement in atherosclerosis. Method: The effects of oxidized low-density lipoprotein (oxLDL) and UCA1 on VSMC proliferation and colony-forming ability was measured by 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, real-time polymerase chain reaction (PCR), and western blots, as well as to determine the effect that oxLDL has on UCA1 expression, and the effect of oxLDL and UCA1 on the expression of cyclin-dependent kinase 2 (CDK2). Results: oxLDL treatment increased the proliferation rate of VSMCs in a concentration-dependent manner. Importantly, UCA1 apparently increased the viability of VSMCs as the VSMCs exhibited a significantly reduced growth rate when UCA1 expression was knocked down by specific small interfering RNAs (siRNAs). In conjunction with increasing cell viability, oxLDL also enhanced the colony-forming ability of VSMCs while UCA1 siRNA decreased the colony-forming ability of VSMCs. Furthermore, UCA1 significantly decreased the percentage of VSMCs in G1 phase, while increasing their percentage in S phase. In contract siRNA knockdown of UCA1 caused an increased percentage of cell in G1 phase, and a reduction in the percentage of cells in S phase. Using real-time PCR and western blot assays, we showed that oxLDL significantly increased the expression levels of UCA1 and CDK2. Furthermore, UCA1 siRNA and CDK2 siRNA almost abolished the positive effect of oxLDL on CDK2 expression. Finally, overexpression of UCA1 induced an increase in CDK2 levels, and knockdown of UCA1 caused inhibition of CDK2 expression. Conclusion: Upregulation of UCA1 enhances abnormal proliferation of VSMC by promoting G1/S transition through modulating the expression of CDK2.

MicroRNA-34c suppresses proliferation of vascular smooth muscle cell via modulating high mobility group box protein 1.

BACKGROUND: Atherosclerosis is the most frequent pathological process that causes cardiovascular diseases. OBJECTIVE: The present study aimed to confirm miRNAs associated with atherosclerosis and explore the molecular mechanism of miR-34c and its target high mobility group box protein 1 (HMGB1) in the control of growth of smooth muscle cells in the development of atherosclerosis. METHODS: Real-time PCR was firstly performed to confirm miRNA correlation with atherosclerosis, and computational analysis and luciferase assay were performed to explore the target of miR-34c, Western blot, and real-time PCR were also utilized to reveal the effect of whether high glucose (HG) and miR-34c affect miR-34c, HMGB1 levels, NF-κB p65 and TNF-α levels, and the role of miR-34c on vascular smooth muscle cells (VSMCs) viability induced by HG. Students' unpaired t test was performed to compare data between two groups. RESULTS: MiR-34c level was associated with atherosclerosis with different expression between VSMCs treated with high glucose or normal VSMCs. Then, HMGB1 is a virtual target of miR-34c with predicted binding site resided in HMGB1 3'UTR and further verified by that miR-34c remarkably reduced luciferase activity of wild HMGB1 3'UTR under a concentration-dependent fashion, and miR-34c cannot influence luciferase activity of mutant HMGB1 3'UTR. CONCLUSIONS: The results suggested miR-34c might be a novel therapeutic strategy in the management of atherosclerosis by suppressing the expression of HGMB1 and its downstream effectors.

A single nucleotide polymorphism located in microRNA-499a causes loss of function resulting in increased expression of osbpl1a and reduced serum HDL level.

Atherosclerosis is the main pathological process that induces CVD (cardiovascular diseases), and the objective of our study was explore whether miR­499a rs3746444 polymorphism was associated with the HDL level, one of the risk factors of atherosclerosis. Online public miRNA database was utilized to predict the miR­499a target, and luciferase assay was conducted to confirm that miR­499a targeted osbpl1a, then western blot analysis and real-time PCR were performed to verify miRNA-mRNA regulatory relationship between miR­499a and osbpl1a. Based on results of bioinformatics algorithms, osbpl1a was predicted as a candidate target gene of miR­499a, luciferase reporter was generated, and it was found that the luciferase activity of cells was substantial downregulated following co-transfection with wild osbpl1a 3'UTR and miR­499a compared to that in scramble control, while the inhibitory effect of miR­499a was abolished after transfection of mutant osbpl1a 3'UTR. Then, miRNA-mRNA regulatory relationship between miR­499a and osbpl1a was detected, a concentration-dependent effect of miR­499a on the miR­499a expression was observed, and both osbpl1a mRNA and protein levels of cells transfected with miR­449a (30 and 60 nM) or osbpl1a siRNA were markedly reduced, while notably improved subsequent to transfect with anti­miR­449a (30 and 60 nM) in comparison with NC groups, moreover, the inhibitory effect among 30 or 60 nM miR­499a, osbpl1a siRNA was similar, the improved effect of 30 or 60 nM anti­miR­499a showed no significant change. The influence of rs3746444 A allele on expression level of miR­499a represented a recessive pattern in high-grade group with a higher level of miR­499a in AA group, and HDL level in AA group was significantly reduced related to those in AG and GG groups. This study validated that rs3746444 polymorphism influenced the expression of miR­499a, its target gene, osbpl1a, and thereby associated with the HDL level, which makes it a potential factor involved in the mechanism of atherosclerosis.