microRNAs inducible by cytokines and regulation of RPE function

National Eye Institute

Investigators

Abstract

Experimental approaches: Human fetal RPE culture (hfRPE) was prepared as described previously and hfRPE was differentiated on transwell of 12-mm diameter inserts for over 4-6 weeks. Experiments were performed using a culture where transepithelial resistance is greater than 200 ohm.cm2. Cytokine cocktail containing TNF alpha, IL-1beta and Interferon gamma were used for the experiment. Total RNA was isolated from control or cytokine treated tissue samples using the mirVana miRNA Isolation Kit (Ambion) according to the manufacturer's instructions. Purified total RNA was quantified using Nanodrop ND1000 (NanoDrop Technologies, Wilmington, DE, USA). Samples for miRNA profiling studies were processed by Asuragen Services (Austin,TX), according to the company's standard procedures from the miRNA-enriched fraction of small RNAs. Relative change of microRNA expression was further determined by the using of real time RT-PCR assay for microRNA (ABI) and fold change was determined by 2-deltadeltaCT method. [unreadable] [unreadable] Results: Approximately 720 microRNAs were detected in normal hfRPE. 595 are human and 121 are derived from Sanger miRbases. By microarray analysis, we identified two microRNAs that were induced to a significant level ( p-value of 0.001 and log2 difference > 1) by the application of cytokine cocktail. Application of interferon gamma didn't produce a significant change in the expression of microRNAs. Subsequent studies of expression levels of these two microRNAs(miR-155, miR-146a) by Taqman quantitative RT-PCR assay revealed that approximately 5-6 fold increase in expression miR-155 ,2-3 fold increase in miR-146a. Future studies will identify molecules or/and pathways that are regulated by these microRNAs.[unreadable] [unreadable] Conclusions: hfRPE is capable of expressing microRNAs that are typical of mediating immunlogical responses. The impact of induction of these microRNAs on RPE physiological function and its pertinence to pathology of age-related macular degeneration will be investigated in the future study.