Alterations of microRNA expression profile and bioinformatics analysis in human microvascular retinal endothelial cell injured by oxidative stress

Abstract

Objective To observe the changes of microRNA(miRNA) expression profile during oxidative stress in human microvascular retinal endothelial cell(HMREC) and to explore the function of the differentially expressed miRNA in the process of retinal vascular endothelial injury. Methods Oxidative stress model was developed with different concentrations of H2O2 in human umbilical vein endothelial cell (HUVEC). Cell viability was determined by using Cell Counting Kit-8 assay. Gene chip technology was used to detect the alterations of miRNA expression profile induced by oxidative stress in HUVEC and bioinformatics analysis was performed in differentially expressed miRNA. Real-time PCR analysis was used to validate the changed miRNA in HMREC. Results According to the results of viability of HUVEC detected by CCK-8 assay, the cell viability of HUVEC treated by 100 μmol/L H2O2 for 8 hours and 16 hours were not different from those of the control group (F100μmol/L=3.897, P>0.05). However, the cell viability of HUVEC treated by other concentrations of H2O2 were decreased significantly and in a dose and time-dependent manner(F200μmol/L=8.172, F800μmol/L=239.214, all P<0.05). The cell viability of HUVEC treated with 400 μmol/L of H2O2 for 24 hours were decreased by nearly 50% (F400μmol/L=6.905, P<0.05). The microarray analysis results showed that there were 116 differentially expressed miRNAs in HUVEC after exposure to H2O2(400 μmol/L) for 24 hours. Among them, 11 miRNAs were decreased or increased for more than 1.5 times. The results of bioinformatics analysis showed that these miRNAs might be involved in multiple biological pathways such as metabolic pathways in cells, adenosine monophosphate activated protein kinase(AMPK) signaling pathway and so on. The results of real-time PCR validated that miRNA-15b, miRNA-106b, and miRNA-497 were significantly downregulated (FmiRNA-15b, HUVEC=9.9, FmiRNA-106b, HUVEC=8.4, FmiRNA-497, HUVEC=63.5, FmiRNA-15b, HMREC=643.7, FmiRNA-106b, HMREC=81.4, FmiRNA-497, HMREC=199.9, all P<0.05), whereas miRNA-195, miRNA-638, miRNA-1246, miRNA-4267, miRNA-4324, and miRNA-4734 were significantly upregulated(FmiRNA-195, HUVEC=592.1, FmiRNA-638, HUVEC=812, FmiRNA-1246, HUVEC=58.5, FmiRNA-4267, HUVEC=1 179.1, FmiRNA-4734, HUVEC=173, FmiRNA-195, HMREC=67.8, FmiRNA-638, HMREC=103.7, FmiRNA-1246, HMREC=2 078.9, FmiRNA-4267, HMREC=234.6, FmiRNA-4734, HMREC=10.7, all P<0.05) in HUVEC and HMREC oxidative stress model treated by 400 μmol/L of H2O2 for 24 hours. Conclusion Oxidative stress induces the imbalance of miRNA expression in HMREC, and differentially expressed miRNA might play a potential role in the process of retinal vascular endothelial injury through AMPK signaling pathway. Key words: Oxidative stress; microRNA; Human umbilical vein endothelial cells; Human microvascular retinal endothelial cells; Bioinformatics analysis