Abstract
BackgroundHigh blood and tissue concentrations of glucose and advanced glycation end-products are believed to play an important role in the development of vascular complications in patients with diabetes mellitus (DM) and chronic kidney disease. MicroRNAs (miRNA) are non-coding RNAs that regulate gene expression in a sequence specific manner. MiRNA are involved in various biological processes and become novel biomarkers, modulators and therapeutic targets for diseases such as cancer, atherosclerosis, and DM. Calcitriol (the active form of vitamin D) may inhibit endothelial proliferation, blunt angiogenesis, and be a cardioprotective agent. Calcitriol deficiency is a risk factor for DM and hypertension. The aim of this project was to study the miRNA microarray expression changes in human umbilical vein endothelial cells (HUVEC) treated in a diabetic-like environment with the addition of calcitriol.MethodsHUVEC were treated for 24 h with 200 μg/ml human serum albumin (HSA) and 100 mg/dl glucose (control group) or 200 μg/ml AGE-HSA, and 250 mg/dl glucose (diabetic-like environment), and physiological concentrations (10-10 mol/l) of calcitriol. miRNA microarray analysis and real time PCR to validate the miRNA expression profile and mRNA target gene expression were carried out.ResultsCompared to control, 31 mature human miRNA were differentially expressed in the presence of a diabetic-like environment. Addition of physiological concentrations of calcitriol revealed 39 differentially expressed mature human miRNA. MiR-181c, miR-15a, miR-20b, miR-411, miR-659, miR-126 and miR-510 were selected for further analysis because they are known to be modified in DM and in other biological disorders. The predicted targets of these miRNA (such as KLF6, KLF9, KLF10, TXNIP and IL8) correspond to molecular and biological processes such as immune and defense responses, signal transduction and regulation of RNA.ConclusionThis study identified novel miRNA in the field of diabetic vasculopathy and might provide new information about the effect of vitamin D on gene regulation induced by a diabetic-like environment. New gene targets that are part of the molecular mechanism and the therapeutic treatment in diabetic vasculopathy are highlighted.
Highlights
MicroRNAs are non-coding RNA species of approximately 22 nucleotides that regulate gene expression in a sequence-specific manner. miRNA join to partial complementary sequences in the 3'UTRs of target mRNA of protein coding genes to specify translational repression and/or mRNA cleavage [1,2]. miRNA are involved in various biological processes and have become novel biomarkers, modulators and therapeutic targets for diseases such as cancer, heart disease, and diabetes [1,2]
Previous studies performed in our laboratory have demonstrated that advanced glycation end products (AGEs) stimulate the endothelial expression of AGE receptor (RAGE) and interleukin 6 (IL6), and depress the eNOS mRNA expression and eNOS enzymatic activity [13,14], while calcitriol blunted the deleterious effect of AGEs in this in vitro model [14]
The addition of calcitriol (10-10 mol/l) to cultured human umbilical vein endothelial cells (HUVEC) exposed to a diabetic-like environment revealed 39 mature human miRNA that were significantly changed (p < 0.05 and fold-change cut-off 1.5), of which miRNA were up-regulated and were down-regulated (Figure 1B)
Summary
MicroRNAs (miRNA) are non-coding RNA species of approximately 22 nucleotides that regulate gene expression in a sequence-specific manner. miRNA join to partial complementary sequences in the 3'UTRs of target mRNA of protein coding genes to specify translational repression and/or mRNA cleavage [1,2]. miRNA are involved in various biological processes and have become novel biomarkers, modulators and therapeutic targets for diseases such as cancer, heart disease, and diabetes [1,2]. Several studies investigated the effect of miRNA on the development of diabetes and its complications, including endothelial and vascular smooth muscle cell dysfunction, diabetic cardiomyopathy and diabetic nephropathy, and demonstrated involvement of specific types of miRNA, which regulate a broad range of inflammatory genes [3,4,5]. High blood and tissue concentrations of glucose and advanced glycation end-products are believed to play an important role in the development of vascular complications in patients with diabetes mellitus (DM) and chronic kidney disease. Calcitriol deficiency is a risk factor for DM and hypertension The aim of this project was to study the miRNA microarray expression changes in human umbilical vein endothelial cells (HUVEC) treated in a diabetic-like environment with the addition of calcitriol
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