s of IL-22 on KC phenotypes Our results reveal that IL22RA1 is regulated by miR-197; moreover, they strongly suggest that IL-22, activates the transcription of miR-197 through STAT3 signaling, thus generating a biochemical feedback loop as summarized in Fig. 5. It was previously shown that IL-22 enhances KC proliferation, increases the thickness PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19674025 of reconstituted human epidermis, inhibits KC differentiation, and enhances KC migration. We asked whether these biological effects of IL-22 will be affected by miR-197 over-expression. To address this question we measured the BrdU incorporation in IL-22 treated HaCaT-miR-197 or HaCaT-HTR, and found it to be significantly buy Digitoxin higher in the HaCaT-HTR cells. In order to evaluate the effect of miR-197 over-expression on IL-22-induced cellular migration, we conducted an in vitro cell migration assay in HaCaT-miR-197 vs. HaCaT-HTR. After seeding, cells were serum starved for 24 h, next IL-22 was added for additional 48 h and cells were fixed. The control HTR-HaCaT migrated to cover 40% of the empty area. The addition of 0.5 or 5 ng/ml IL-22 in the serum free medium led to an increase in the covered area of 52% and 56%, respectively, signifying IL-22-iduced motility. HaCaT-miR-197 had a significantly lower level of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19673983 baseline migration, covering only 510% of the empty area at 48 h, without any significant change in migration following IL-22 treatment. In parallel we treated HaCAT with antago-miR-197, in the presence or absence of IL-22. Our results suggest that the antagomiR-197 had no significant effect on KC proliferation. The miRNAs role as “fine-tuner”might explain the fact that in high over expression of repressor miRNA we were able to induce 4 Crosstalk between IL-22 Signaling and miR-197 5 Crosstalk between IL-22 Signaling and miR-197 biological effects, however down regulation of one out of many “fine tuners”had no biological effect. DNA methylation of the miR-197 putative promoter regions ical feedback loop. However, despite the high levels of IL-22 in the blood of psoriatic patients and high expression of IL22RA1 in psoriatic patients’ skin, the expression of miR-197 is decreased in their KC. Recent work suggest that DNA methylation in the skin is dynamic and it changes along the epidermal layers and in specific genes. DNA methylation is capable of regulating both gene repression and activation, and the basal status of promoter methylation is important for individual genes expression. A recent study comparing differences in the DNA methylation, between psoriatic lesions and uninvolved or normal skin revealed Crosstalk between IL-22 Signaling and miR-197 many CpG sites with differential methylation levels. We hypothesized that cytosine methylation of the miR-197 putative promoter in psoriatic lesion compare to normal skin might be different and thereby explain why miR-197 is silenced in psoriasis despite the high levels of IL-22 in the patients’ blood. The,2000 bases up stream of the pre-miR-197 sequences comprise the regulatory elements of promoter and contain one CpG island. We analyzed the DNA methylation of this region in biopsies from formalin fixed paraffin embedded, of psoriatic lesions, uninvolved psoriatic skin and normal skin. Each sample was subjected to at least two sequencing analyses. We can see that the methylation pattern of the mapped CpG island in the miR-197 putative promoter, in psoriatic samples is similar to normal skin. Discussion Recently we have shown that miR-197 expressio