Expression Of MiR-486-3p In Psoriasis And Its Inhibition On The Keratin17and The Effect In Pathogenesis Of Psoriasis

Psoriasis is an easily-recurrent chronic inflammatory skin disease. The pathogenesisof psoriasis is not fully understood. It is now considered as a skin disease caused byenvironmental and genetic factor that make innate and adaptive immune dysfunction. Th1and Th17cells and the cytokines secreted by them play an important role in thepathogenesis of psoriasis.Pathological changes of psoriasis is due to the fast proliferation of keratinocytes andthe abnormal KCs express high level of keratin17, but the expression of K17is low innormal skin. K17is highly expressed in the tissue surrounding healing of wound. Ourprevious study found that the circulation pathway K17autoreactive T cells cytokineslead to the occurrence and maintenance of psoriasis. A variety of cytokines can regulatethe expression of K17, but the mechanism of its post-transcriptional regulation is not clear.MiRNA,as one of the important post-transcriptional regulators, are involved inmany biological activities,such as cell proliferation,differentiation and migration, etc. Ourstudy found that miR-486-3p may be contributed to the inhibition of post-transcription ofK17and influence the proliferation and migration of KC in psoriasis throughbio-informatics prediction and literature review, which improved the current knowledge ofK17in pathogenesis of psoriasis and provide new strategies for the K17as a target in the treatment of psoriasis.Objective: Investigation the mechanism of post transcriptional regulation of K17,verification the function of the molecular regulating K17in psoriasis and furtherexploration its effect on KCs.Methods: Screen the miRNA may be involved in the post-transcriptional regulation ofK17through bio-informatics prediction and literature review, then conduct clinicalsamples verification by the qRT-PCR technique to confirm the screened miRNAsfunction. K17protein was detected by Western Blot on HaCaT cells by overexpression ofmiR-486-3p and then RT-PCR used to detect the expression of K17mRNA. Constructionof plasmids contained K173UTR region s seed sequence, seed sequence deletedfragment of MUT-1,MUT-2which is point mutation fragment of the seed sequence andMUT-3which is repeated fragment of the seed sequence. Then detect the inhibitionmechanism of miR-486-3p on the expression of K17through Dual luciferase reportersystem. Adopt CCK8checking the variation of proliferation of over-expressed K17HaCaT cells, which are transfected by miR-486-3p analogues, also observe the changes ofthe migration ability through cell scratch test.Results:8bases showed by the results of bio-informatics prediction on miR-486-3p cancombine with the seed sequence of3UTR region of K17, and based on the results of genechip from literature review, the expression of miR-486-3p are39%higher in the psoriaticlesion than in the normal skin. During the results of the level of miR-486-3p expressiondetected through RT-PCR between10cases of patients with psoriatic lesions and10healthy controls,0.211±0.120was obtained in the psoriatic lesions which is lower thanthe healthy controls results of0.555±0.425,and it is0.380than the healthy control,（t=2.616，υ=9，p<0.05）；According to the results of Western Blot,the obvious decreasedexpression level of K17can be found in the transfected miR-486-3p analogues of HaCaTcells. The fluorescein intensities inside two groups of constructed plasmids were65.31±6.32and54.18±10.01respective in contained K173UTR region s seed sequence andMut3,which are lower than control group（p<0.05），and the results of Mut1and Mut2were114.77±16.14and110.21±12.99,no obvious difference with control group （p>0.05）；The results of CCK8detected cell proliferation implicated that the absorbancevalue of the group transfected mimics is1.005±0.099and the transfected NC fragment sgroup is1.235±0.119，(t=2.860， p<0.05)； The speed of migration slows downsignificantly in the over-expressed K17HaCaT cells transfected mimics than thetransfected NC fragment s group, which observed by cell scratch test.Conclusion: The expression level of miR-486-3p was significantly decreased in thelesions of psoriasis than in normal skin, which can induce increase of K17expression. Themechanism of above results comes into play through the combination of miR-486-3p withthe3UTR region s seed sequence of K17. The inhibition of K17by miR-486-3p cansuppress the proliferation and migration of keratinocytes.