miR-96 impress in INS/AKT/GLUT4 axis signaling in mRPE cells
Narges Zolfaghari1 , Zahra-Soheila Soheili1 *, Shahram Samiei2 , Ali Hafezi-Moghadam3 , Maliheh Davari1 , Ehsan Ranaei-Pirmardan3 , Hamid Ahmadieh4 , Zeynab Hosseinpoor1
- Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
- Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
- Molecular Biomarkers Nano-Imaging Laboratory, Brigham and Women's Hospital, Boston, MA, USA
- 4. Ophthalmic Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Abstract: Diabetic retinopathy is the leading cause of blindness in working-age population. INS/AKT/GLUT4 axis is the most important cell signaling pathway in the uptake and consumption of glucose by the exposed cells. New findings suggest that many abnormal microRNAs expressions are involved in the pathogenesis of DR. We investigated the role of miR-96 in INS/AKT/GLUT4 axis signaling pathway in mRPE cells.
Methods: mmu-miR-96 gene was synthesized and sub-cloned into the pAAV2-MSC-eGFP vector. mRPE cells were transfected with the corresponding vector using calcium phosphate precipitation method. To search for the role of miR-96, 30nM of Antagomir-96 or 30nM of scrambled molecules were transfected in miR-96 overexpressed mPRE cells using lipofectamine 2000. Total RNA was isolated from treated mRPE and control cultures and interested genes’ expression was quantified using RT-qPCR. MTT and scratch wound healing assays were performed to investigate miR-96 role in proliferation and migration of mRPE cells.
Results: The mmu-miR-96-5p gene was successfully sub-cloned. MTT assay showed that, miR-96 reduced proliferation of mRPE cells. Wound-healing assay results showed that, under miR-96 overexpression and miR-96/Scrambled molecules overexpressed mRPE cultures the mobility of mRPE cells was significantly increased (wound closure rate 21.23% and 25.5%, respectively). |However, the wound closure rate of untreated GFP expression and, miR-96/Antagomir-96 overexpressed mRPE cells were not significant after 24h. miR-96 relative expression in miR-96 overexpressed mRPE cells was increased 13.7 fold in comparison to GFP overexpressed cells. RT-qPCR results for PIK3CA, Pak1, Snap23 and, AKT2 showed 0.1, 0.51, 0.07 and, 0.42 fold reduction in miR-96 treated cells compared to control. FOXO1 showed 5.43 fold increased level in treated samples. The relative gene expression in miR-96/Antagomir-96 transfected mRPE cells compared to miR-96/scramble transfected cells represents that, PIK3CA, Pak1, Snap23, AKT2 and, FOXO1 expression changed to 1.7, 3.03, 5.88, 3.37 and, 0.48 fold respectively.
Conclusion: Previous studies had reported that, mmu-miR-96 was upregulated in the retina of diabetic animals. However, the mechanism of its function is still unknown. Overexpression of mmu-miR-96-5p showed a reduction in cell proliferation and an increase in cell mobility. On the other hand, miR-96 interfered with the expression of genes involved in INS/AKT/GLUT4 signaling pathway, which were confirmed by suppression of miRNA-96 using Antagomir-96.
