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Caroline E. Haldin, F F. Costa, E F. Vanin, V J. Dudley, A C. Arman, A P. Sampath, M B. Soares, V P. Sarthy; miRNA Profiling Of Müller Glial Cells During Retinal Degeneration. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5357.
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microRNAs (miRNAs) are small non-coding RNAs (21-25nt) that regulate gene function by targeting specific sequences within the mRNA thus causing a reduction in translation efficiency or degradation of the mRNA. Several miRNAs have been previously identified as highly expressed specifically in the retina, and during retina degeneration. This study aims to examine the roles of miRNAs in retina degeneration, focusing on those highly expressed in gliotic Müller cells.
eGFP-expressing Müller cells were isolated from retinas of wild type or rd1 (retinal degeneration) mice by fluorescence-activated cell sorting (FACS) at P14, P35 and P50. Total RNA was extracted using the MirVana isolation kit and mature miRNA cDNA was synthesized using Megaplex Primer Pools with preamplification. Rodent Taqman low-density arrays (TLDA) were used to profile 585 miRNAs including well characterized, more recently discovered and mir* sequences, on a 7900HT Fast Real-Time PCR System controlled by the Sequence Detection Systems (SDS) and Relative Quantification (RQ Manager) software. Statistical analysis was carried out using StatMiner software.
Using a nine-fold change cut-off limit at P14, the peak of rod degeneration, four miRNAs were identified that had significantly increased expression levels in the rd1 Müller cells, mmu-mir292-3p (42.46 fold), mmu-mir-677 (18.6 fold), rno-mir-450a (12.44 fold) and mmu-mir-296-3p (11.93 fold). Three of these miRNAs have predicted target sequences in several genes that we have identified by qPCR as being down regulated in rd1 Müller cells. Interestingly, in each of the down-regulated genes, at least two, if not all three of the miRNA’s target sequences are predicted. Currently, the miRNAs expression levels and localization are being confirmed using Taqman qPCR and in situ hybridization, target sequence and function is being validated by reporter constructs and inhibition assays, and data from P35 (the start of cone degeneration) and P50 (during cone degeneration) are being compared with that from P14.
Results from the P14 analysis indicate that there are changes in Müller cell miRNA expression levels during gliosis that likely play an important role in the regulation of mRNAs during this process. To date, miRNA expression has only been investigated in the whole retina in models of RD and RP, therefore our work highlights the importance of studying individual cell types within the retina in order to get a clearer picture of gene function and hierarchy.
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