mmu-miR-96 Expression In mRPE Cell Line Under High Glucose Condition

Narges Zolfaghari1 , Zahra-Soheila Soheili1 *, Maliheh Davari1 , Shahram Samiei2

  1. Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
  2. Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

Abstract: Diabetic retinopathy (DR) is one of the most common cause of visual impairment / blindness which is associated with duration of diabetes. There are treatments for DR, but most of them target the advanced stages of the disease, show remarkable side effects and many patients do not respond to the treatment regimen as much as necessary. Recent reports suggest that miRNA expression profile is dysregulated during pathogenicity of DR. So, therapeutic role is plausible for miRNAs in diabetic retinopathy. To understand the mechanisms by which miR-96 is involved in diabetic retinopathy, we studied the cluster miRNA including miRNA-96, miRNA-182, and miRNA-183.

Methods: mRPE cell line was seeded in DMEM-F12 culture medium supplemented with 10% FBS and penicillin and streptomycin. The cultures incubated at 37 °C in a humidified atmosphere with 5% CO2. The next day the cells were treated with normal glucose (5 mM) or high glucose (30 mM) or similar concentrations of mannitol -which applied as osmotic control- for 24 h. Total RNA was extracted using TriPure isolation reagent. Stem loop Real-Time PCR was used to evaluate the expression level of mmu-miR-96 gene. MTT assay determined the viability of the cultures.

Results: The relative expression of mmu-miR-96 was calculated using the 2−ΔΔCt method and RNU48 served as the endogenous control. Relative expression of miR-96 in 5 mM glucose respected to the 5 mM mannitol samples was 0.74. While, under the treatment of the 30 mM glucose 3.89-fold increase was detected respected to mannitol treated cultures. MTT assay showed no difference in cell viability at different concentrations of glucose and mannitol.

Conclusion: Previous studies had reported that mmu-miR-96 was upregulated in STZ induced diabetes rat’s retina. However the mechanism of its function is still unknown. We intended to identify the role of miR-96 in high glucose condition, in vitro which mimics DR condition. The result of stem loop Real-Time PCR showed that, miR-96 increased in high glucose condition. The next step is to measure the expression of target genes of miR-96 in the treated samples to understand the mechanism of miR-96 function in high glucose concentration.





اخبــار



برگزار کنندگان کنگره


حامیان کنگره