| Objective: To explore the differentially expressed miRNA of human retinal capillary endothelial cells(HRCECs) in hyperglycemic environment by miRNA gene chip, preliminary screen the miRNA which associated with diabetic retinopathy(DR), then adopt bioinformatics method to forecast target genes of part differentially expressed miRNA. This is useful for further study on miRNA role in pathogenesis of DR and provide theoretical basis for DR prevention and treatment. Methods: 1. Cultured the HRCECs in DMEM(high glucose) medium which including 10% fetal bovine serum, 50mg/L streptomycin and 50mg/L penicillin, put them in 5%CO2 incubator. 2. Took the 3-4 generation growth good cells and implanted them in six orifice plate, added D-glucose or mannitol in DMEM medium to made into conditioned medium, divided the cells into three groups: a. normal control group: DMEM medium with 25mmol/L glucose; b. high glucose group: conditioned medium with 90mmol/L glucose; c. mannitol high permeability control group: conditioned medium with 65mmol/L mannitol and 25mmol/L glucose. 3. each group cells were cultured in the above conditions for five days, then used In Situ Cell Death Detection Kit for apoptosis detection; total RNA of normal control group and high glucose group was isolated by RNeasy Mini Kit, ultraviolet spectrophotometer detected total RNA purity and concentration, formaldehyde degeneration agarose gel electrophoresis evaluated its purity and integrity. 4. Used miRCURY Array Power Labling kit to prepare fluorescent marker probe, labeled with Hy3, the miRNA were hybridized on miRNA array under standard condition. Hybrid images were scanned by GenePix 4000B microarray scanner, digital conversion of hybrid images and calculation of standard values by GenePix pro V6.0, Part results of miRNA array were verified by real-time quantitative polymerase chain reaction (PCR), used the softwares of target genes prediction such as DIANA-microT, PicTar and TargetScan to predict targets, and selected target genes which at least appeared in two softwares. Results: 1. Used inverted microscope to observe HRCECs: HRCECs were monolayer adherent cells and presented a similar appearance of pebbles. Observed apoptotic HRCECs 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. This demonstrated that high glucose can induce apoptosis in HRCECs, ? and the effects of high glucose were not because of medium by high molarity osmotic pressure. 2.Quality testing of total RNA: with spectrophotometer measurement, the ratio of absorbance of total RNA in normal control group atA260/A280nm was 1.99, at A260/A230 was 2.05; total RNA of high glucose group at A260/A280 was 1.98, at A260/A230 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. 3. The results of miRNA gene chip showed that: compared with normal control group, a total of 49 miRNAs were differentially expressed in high glucose group (fold change>2 or fold change <0.5), including 31 up regulated and 18 down regulated. 4. We used quantitative RT-PCR to confirm the expression level of has-miR-320c and has-miR-29a*, the results were consistent with the chip. ?The prediction of target genes showed that their targets were widely, which involved many growth factors and proteins. Conclusions: 1.We found the differentially expressed miRNA of human retinal capillary endothelial cells in hyperglycemic environment, the results indicate that the abnormal expression of miRNA may correlate to pathogenesis of Diabetic retinopathy. 2. The results of quantitative RT-PCR in has-miR-320c and has-miR-29a* are consistent with miRNA array, which shows the results of miRNA array are true and correct. |
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