Study on the differentially expressed microRNA of human retinal microvascular endothelial cells in high glucose environment by microRNA gene chip

Abstract

To explore the differentially expressed microRNA (miRNA) of human retinal microvascular endothelial cell (HRCEC) in hyperglycemic environment by miRNA gene chip, then adopt bioinformatics methods to forecast target genes of part differentially expressed miRNA. Experimental study. HRCEC were cultured. Took the 3-4 generation growth good cells and divided the cells into three groups: (1) normal control group: DMEM medium with 25 mmol/L glucose; (2) high glucose group: conditioned medium with 90 mmol/L glucose; (3) mannitol high permeability control group: conditioned medium with 65 mmol/L mannitol and 25 mmol/L glucose. Each group cells were cultured in the above conditions for five days, then used in situ cell death detection kit for apoptosis detection; the total RNA was isolated and examined; the differentially expressed miRNA were detected by miRNA gene chip, part results of miRNA array were verified by real-time quantitate polymerase chain reaction (PCR), potential miRNA targets were analyzed by bioinformatics methods. Observed apoptotic HRCEC by fluorescence microscope: the nucleus of normal control group and mannitol control group were dyed by DAPI and appeared blue fluorescence, but hadn't apoptosis fluorescent signals; the nucleus of high glucose group also appeared blue fluorescence, and had green apoptosis fluorescent signals. Quality testing of total RNA: with spectrophotometer measurement, the ratio of absorbance of total RNA in normal control group at A(260)/A(280) nm was 1.99, at A(260)/A(230) was 2.05;total RNA of high glucose group at A(260)/A(280) was 1.98, at A(260)/A(230) was 2.26. The results of formaldehyde degeneration agarose gel electrophoresis showed that the electrophoresis strips were clear and complete, indicated that the total RNA had better quality and high purity. Compared with normal control group, 49 miRNAs were found to be differentially expressed in high glucose group (fold change > 2 and fold change < 0.5), including 31 up-regulated miRNAs and 18 down-regulated miRNAs. The results of real-time quantity PCR revealed that hsa-miR-320c and hsa-miR-29a(*) were up-regulated in high glucose group, which were consistence with the miRNA gene chip. Furthermore, the target genes prediction of two above miRNAs were involved many growth factors and proteins. miRNA are differently expressed in HRCEC under hyperglycemic conditions.