Role Of MicroRNAs In The Pathogenesis Of Metabolic Memory

Objective:To establish the model of "metabolic memory" on rat aortas and human aortic endothelial cells (HAECs), and observe the metabolic indices in rats, the nuclear transcription factor-κB (NF-κB) p65subunit and pro-inflammatory gene in "metabolic memory".Methods:1. All the rats were randomly categorized into3groups,1) normal, with normal blood glucose for24weeks;2) insulin treated diabetic, with hyperglycemic for12weeks and then good glycemic control with insulin during the following12weeks; and3) diabetic, with poor glycemic control for24weeks.2. HAECs were divided into4groups,1) normal control, exposed to normal glucose (5mmol/L) for7days;2) osmotic control, exposed to30mmol/L mannitol for7days;3) metabolic memory, exposed to high glucose for1day and followed by6days of normal glucose; and4) high glucose, exposed to high glucose (30mmol/L) for7days.3. The mRNA expression levels of p65, monocyte chemotactic protein-1(MCP-1), and interleukin-6(IL-6) in the rat aortas and HAECs were detected by real-time polymerase chain reaction (PCR); the protein expression levels of p65were detected by Western blot; and stem-loop real-time PCR was used to detect the expression of miR-146a levels.Results:1. The normal control group rats have weight increasing, metabolic memory and high glucose group after injection of STZ rats’weight gain rate is lower than the normal control group; Metabolic memory group rats’weight growth rate is higher than the high glucose group, lower than the normal control group. 2. The expression of p65, MCP-1, and IL-6was significant increased in the model of "metabolic memory" on rat aortas and HAECs.Conclusion:1. Animal and cell models of "metabolic memory" are successfully established.2. The expression of p65, MCP-1, and IL-6is significant increased, and NF-κB pathway is persistent activated in "metabolic memory" Objective:Respectively to observe expression of the miR-146a and miR-484in hyperglycemia "metabolic memory" state, through bioinformatics method to find and determine their target genes, observe the target genes expression in hyperglycemia "metabolic memory" state.Methods:1. Stem-loop real-time PCR was used to detect the expression of miR-146a and miR-484levels in the rat aortas and HAECs.2. The target gene prediction softwares were used to predicte target genes of miR-146a and miR-484. The changes of predicted target protein level in the rat aortas and HAECs were detected.Results:1. The expression level of miR-146a was significant decreased in the model of "metabolic memory".2. The expression of TNF receptor-associated factor6(TRAF6) and interleukin-1receptor-associated kinase1(IRAK1) which miR-146a was predicted to base-pair with sequences in the3’-untranslated regions (UTRs), was significant increased in the model of "metabolic memory".3. The expression level of miR-484was significant decreased in the model of "metabolic memory".4. The expression of vascular endothelial growth factor B (VEGFB) which miR-484was predicted to base-pair with sequences in the3’-untranslated regions (UTRs), was significant increased in the model of "metabolic memory".Conclusion: 1. MiR-146a is significant decreased and its target genes IRAK and TRAF6are significant increased in "metabolic memory".2. MiR-484is significant decreased and its target genes VEGFB are significant increased in "metabolic memory". Objective:To validate the effect of miR-484and miR-146a on nuclear transcription factor-KB (NF-κB) p65subunit, pro-inflammatory gene and its functional targets in the model of "metabolic memory" on rat aortas and human aortic endothelial cells (HAECs).Methods:1. The changes of p65, MCP-1, and IL-6in HAECs were detected after miR-146a mimics/inhibitors transfection. The changes of predicted target protein level in the rat aortas and HAECs were detected before and after miR-146a transfection.2. The expression levels of p65, MCP-1, and IL-6were detected after miR-484mimics/inhibitors transfection. The change of VEGFB was detected after miR-484mimics/inhibitors transfection.3. The changes of p65, MCP-1, and IL-6were detected after VEGFB over-expression plasmid or siRNA transfection.Results:1. The expression of p65, MCP-1, and IL-6was significant decreased after miR-146a mimics transfection; and conversely was increased after miR-146a inhibitors transfection.2. Inhibition of miR-146a increased the expression of TRAF6and IRAK1, and miR-146a mimics led to the decrease of TRAF6and IRAK1.3. Inhibition of miR-484increased the expression of p65, MCP-1, and IL-6; and miR-484mimics led to the decrease of p65, MCP-1, and IL-6.4. Inhibition of miR-484increased the expression of VEGFB; and miR-484mimics led to the decrease of VEGFB. 5. The expression of NF-κB p65, MCP-1, and IL-6was significant increased after VEGFB over-expression plasmid transfection; and conversely was decreased after VEGFB siRNA transfection.Conclusion:1. MiR-146a is significant decreased in "metabolic memory", and could negative regulat NF-κB pathway by IRAK and TRAF6.2. MiR-484is significant decreased in "metabolic memory", and could negative regulat NF-κB pathway by VEGFB.