Study On The Roles Of RNA N6-methyladenosine Methylation In Psoriasis Vulgaris

Background:Psoriasis is a chronic,systemic,immune-mediated disease^[1]).The most frequent phenotype is psoriasis vulgaris(PsV),accounting for 80-90%of all subtypes,usually triggered by trauma,infection,and drugs in genetically susceptible populations^[1].The hallmark histopathological features of psoriasis vulgaris are epidermal acanthosis(thickening of viable layers),hyperkeratosis,and parakeratosis,accompanied by inflammatory infiltrate in the dermis^[2].The complex interactions between keratinocytes and T cells are responsible for the development and maintenance of psoriatic inflammation^[3].In recent years,with the emergence of antibody-based,high-throughput sequencing,namely,methylated RNA immunoprecipitation with next-generation sequencing(MeRIP-Seq),m⁶A methylation has become a hotspot of scientific research,in which unprecedented progress has been achieved.Among the various types,N6-methyladenosine(m⁶A)methylation is the most common internal modification in higher eukaryotic messenger RNA(mRNA)and long noncoding RNA(LncRNA).m⁶A methylation is also the most pervasive and deeply studied mRNA modification,and plays an important role in the process of affecting cell fate and function^[4].m⁶A methylation is dynamic and reversible,which is determined by methyltransferases("writers"),demethylases("erasers"),and binding proteins("readers").The biological functions of RNA m⁶A methylations occur at three different levels:molecular,cellular,and physiological.At the molecular-level,m⁶A methylations posttranscriptionally regulates RNA splicing,transport,translation,stability,and localization^[5].At the cellular level,m⁶A methylations determine the fate of mammalian embryonic stem cells(mESCs)and play a powerful and precise regulatory role in cell proliferation,differentiation and development programmes^[6].At the physiological level,reversible m⁶A methylation has various consequences^[7,8].These processes are closely related to the pathogenesis of psoriasis vulgaris as mentioned above.Studies of aberrant m⁶A in human diseases are rapidly evolving,such as in cancer,obesity,infertility,neuronal disorders,immune diseases,and inflammation^[33-37].However,the regulatory mechanism and physiological significance of m⁶A methylation in psoriasis vulgaris are still poorly understood.Objective:Assess the level of m⁶A methylation and its related enzymes in the skin lesions of patients with chronic plaque psoriasis(psoriasis vulgaris)and analyzed the correlation.Establish a IMQ-induced psoriasis-like mouse model and a Mett13 heterozygous knockout mouse model to detect the effect of METTL3 mediated m⁶A methylation on the development of psoriasis vulgaris.Finally,MeRIP-seq was conducted to identify the transcripts of m⁶A methylation in psoriasis vulgaris.Methods:First,20 patients with PsV and 20 healthy controls were randomly selected to measure m⁶A level in total RNA of skin lesions by m⁶A RNA methylation quantification kit.The expression levels of methylation related enzymes were evaluated by quantitative reverse transcription PCR(RT-qPCR).The correlation between the two groups was analyzed.Second,a IMQ-induced psoriasis-like mouse model and a Mettl3 heterozygous knockout mouse model were established.The effects of METTL3 mediated m⁶A methylation on psoriasis vulgaris phenotype,epidermal thickness,Thl and Th17 cell ratio,inflammatory cell infiltration and inflammatory factor expression were evaluated by psoriasis area severity index(PASI),RT-qPCR,Western blot(WB),immunohistochemistry(IHC)and flow cytometry(FCM).Third,MeRIP-seq was conducted to identify the transcripts of m⁶A methylation in psoriasis vulgaris.Results:The level of m⁶A methylation and the methyltransferase METTL3 in psoriatic lesions were significantly lower than those in healthy controls.There was a significant positive correlation between the level of METTL3 and m⁶A methylation in psoriatic lesions.The level of m⁶A methylation and METTL3 in psoriatic lesions were both negatively correlated with PASI score.In addition,consistent with the results of human samples,the expression level of METTL3 in the skin lesions of IMQ-induced psoriasis-like mice was significantly lower than that of healthy control mice,and the levels of METTL3 in the epidermis and dermis of IMQ group were both lower than that of healthy control mice.Heterozygous knockout Mettl33 in IMQ-induced psoriasis-like mouse model down regulated the m⁶A methylation and aggravated the development of psoriasis vulgaris,including aggravating the skin lesion phenotype,epidermal thickness,the proportion of Thl and Th17 cells,the degree of inflammatory cell infiltration and the level of inflammatory factors.Finally,MeRIP-seq showed that the transcripts contained down regulated m⁶A methylation were highly enriched in Wnt signaling pathway.Conclusion:The level of m⁶A methylation and its catalytic enzyme METTL3 in psoriasis vulgaris were significantly down regulated in psoriasis vulgaris.Down regulated METTL3 mediates the down-regulation of m⁶A methylation,which aggravates the occurrence and development of psoriasis vulgaris.The down-regulation of m⁶A methylation may aggravate the development of psoriasis vulgaris by regulating Wnt signaling pathway.