Effect Of1,25-dihydroxyvitamin D3on MicroRNAs Profiling In HaCaT Cell

Objective:To identify the regulation of1,25-dihydroxyvitamin D3on microRNAs (miRNAs) profiling in human keratinocyte HaCaT cell line and screen the targets of1,25-dihydroxyvitamin D3. To make a foundation for demonstrating the mechanisms of1,25-dihydroxyvitamin D3therapy for psoriasis.Methods:Transfer1×105cells/well in a6-well plate. HaCaT cells were cultured in DMEM medium with10%fetal serum and incubated in a humidified37℃,5%CO2. HaCaT cells were treated by10-6M1,25-dihydroxyvitamin D3when the confluence of cell is more than70%. After48hours, cells were harvested and total RNA was isolated. And then we screened the miRNAs profiling in the HaCaT cells treated by1,25-dihydroxyvitamin D3and negative control using miRCURYTM LNA expression array (Exiqon). MiRNAs with significant changes were analyzed and confirmed by realtime PCR.Results:According to the results from miRNAs array, we found that89miRNAs with no less than3-fold change were up-regulated and43miRNAs were down-regulated in HaCaT cells treated by1,25-dihydroxyvitamin D3compared with negative control (Fold Change cut-off:3.0). The top10hsa-miR-340-3pmiRNAs up-regulated in treatment group includes, hsa-miR-2113, hsa-miR-550b-3p, hsa-miR-520d-5p, ebv-miR-BART18-3p, hsa-miR-374c-3p, hsa-miR-4292, hsa-miR-488-3p, hsa-miR-221-5p, hsa-miR-668. The top10miRNAs down-regulated in treatment group includes hsa-miR-331-3p, hsa-miR-429, hsa-miR-15b-5p, hsa-miR-30c-1-3p, hsa-miR-31-3p, hsa-miR-3653, hsa-miR-182-5p, hsa-miR-339-5p, hsa-miR-181d, hsa-miR-194-3p. Furthermore, we chose three miRNAs (miR-99b-5p, miR-222-3p and miR-205-5p) for validating the results of microarray using realtime PCR, which were expressed aberrantly in lesional skin of psoriasis with more than3-fold changes between treatment and controls. The results of realtime PCR showed that miR-99b-5b expression was up-regulated and expression of miR-222-3p and miR-205-5p was down-regulated in1,25-dihydroxyvitamin D3treated cells compared with negative controls, which is concordance with the results of microarray. So the results from our miRNAs expresson profiling are reliable.Conclusions:1,25-dihydroxyvitamin D3can regulate many miRNAs expression in HaCaT cell. miR-99b-5p, miR-222-3p and miR-205-5p may be the potential targets of1,25-dihydroxyvitamin D3.