Expression Of MicroRNA-210in Skin Lesion Of Psoriasis Vulgaris And Its Effect On Keratinocytes

ObjectiveTo compare miR-210expression levels in skin lesions of psoriasis vulgaris. To predict and verify target genes of miR-210. To study protein expression of RUNX3in skin lesions of psoriasis vulgaris. To study the influence of miR-210on RUNX3protein, cell proliferation, apoptosis, and cellcycle in HacaT cells.MethodsLesional skin of20cases of patients with vulgaris psoriasis and normal skin of16cases of normal controls was separated. TRIzol reagent was used to extract total RNA.,Protein extract kit was used to isolate total protein. Real-time PCR was used to derminate miRNA-210expression levels in20cases of patients with psoriasis vulgaris and16cases of healthy controls. western blot was used to derminate target gene protern expression levels. Using Mirbase/Targetscans/PicTar target gene prediction software to find target genes of miR-210, we selected RUNX3as target gene, which may be closely related with the onset of psoriasis. Moreover,we constructed the wild-type and mutant-type luciferase reporter plasmids, which contain either the wild-type putative miR-210binding sequence (RUNX3WT-luciferase), or the miR-210binding sequence containing two point mutations (RUNX3Mut-luciferase) in the3-UTR region of RUNX3. The plasmids were then co-transfected into HacaT cells with miR-210mimic and miR-210mimic control by lipofectamine2000. After48hours firefly luciferase activity was measured using the Dual-Luciferase reporter assay system and luminometer. Renilla luciferase was used as internal control. miR-210inhibitor and negative control (miR-210inhibitor control) were tranfected into HacaT cells by lipofectamine2000. Real-time RT-PCR method was to detect the expression of miR-210, Western blot was used to detect RUNX3protein expression level, and MTT method was used to detect cell proliferation. Cell apoptosis and cell cycle were detected by Flow cytometry instrument.Results1. Real-time PCR results showed that expression level of miR210was decreased in the lesions of patients with psoriasis vulgaris group compared with normal control group (p<0.05). Compared with healthy controls, RUNX3protein expression level was increased significantly in the skin lesion of patients with psoriasis vulgaris (P＜0.05)2. RUNX3is a predicted target of miR-210according to the bioinformatic algorithm software. Co-transfection of miR-210mimic and RUNX3WT-luciferase in HacaT cells can inhibit the expression of RUNX3WT-luciferase activity (p<0.05), but failed to inhibit RUNX3Mut-luciferase activity.3. In the transfected HacaT cells with miR-210inhibitor relative to miR-210inibitor control group, miR-210expression level was decreased and RUNX3protein expression levels increased significantly (P<0.05). Furthermore, we found that cell viability of HacaT cells transfected with miR-210inhibitor was increased and the rate of apoptosis was decreased signfiicantly (P<0.05). However, no significant difference was observed in cell cycle (P>0.05).Conclusion1. The expression level of miR-210was down-regulated in skin lesions of patients withpsoriasis vulgaris.2. RUNX3is the target gene of miR-210which can inhibit protein expression of RUNX3by binding to the3’-UTR of RUNX3.3. Decreased miR-210expression in HacaT cells contribute to the increased cell vitality and down-regulated rate of cell apoptosis.