In these studies, we used a previously established primary culture model of hfRPE shown to be responsive to stimulation by an inflammatory cytokine mixture (ICM) consisting of TNF-, IL-1, and IFN-. Observed responses include a decrease in transepithelial resistance (TER), an increase in fluid absorption, polarized secretion of angiostatic/angiogenic cytokines, and changes in miRNA expression levels. By using the Asuragen DiscovArray miRNA Expression Service, we measured the expression levels of l 13,000 confirmed and putative miRNAs and found that only miR-155 was significantly increased by ICM. Transfection of a miR-155 mimic into intact monolayers of hfRPE significantly decreased TER to l 60% of control (850 b 160 cm2;mean bSD);a similar result was previously obtained by addition of ICM. This result strongly suggests that the effects of pro-inflammatory cytokines are in part determined by miR-155. In order to study these effects, changes in mRNA expression in the ICM treated and miR-155 mimic transfected hfRPE were studied using Affymetrix gene expression array. We found a total of 585 genes, common to both groups, which had similar expression pattern changes relative to control. A scatter plot showed that the change in both groups was highly correlated. Using Ingenuity Pathway Analysis (IPA), we identified components of several canonical signaling pathways (IFN and NFkB) that are expected to be involved in ICM signaling and a subset of genes (e.g., APC, CLCN5, CSF1R, LRAT, PCDHB5, SLC13A3, JAK2, SOSC1), identified as in silico targets of miR-155, were critical for ocular function. This data suggest that miR-155 may directly impact on ICM-induced signaling cascade and affect hfRPE physiological function through its target genes. To carry out these experiments, we used a Human Phospho-kinase antibody array to measure the phosphorylation levels of forty-six phospho-kinases simultaneously. We found several kinases, and changes in their phosphorylation levels were detectable by both miR-155 and ICM (e.g., SRC, AKT, B-catenin, and p53). Using IPA, we identified a predominant change in SRC phosphorylation and this change lies upstream of the other kinases. Thus, identification of the miR-155 target genes in regulating SRC phosphorylation will be crucial in determining miR-155 function.
