KMT2C Affects Keratinocyte Hyperproliferation And Psoriasiform Skin Inflammation Via Upregulating Transcription

BackgroundPsoriasis,an immune-mediated chronic inflammatory disease in the skin,nails and joints,affects 2%to 4%of people worldwide.The patient’s skin lesion is characterized by silver-kind scaly erythema,plaques.The disease has a long course and is difficult to cure,easy to relapse,so it profoundly impairs their quality of life.At present,it is widely accepted that the etiology of psoriasis involves genetic susceptibility and environmental that make keratinocytes interacted with the Th cells and their secreted cytokines,which leads to abnormal keratinocyte proliferation,differentiation.Various factors,including various cells,immune molecules,cytokines,chemokines,and intracellular signaling pathways,are involved in this pathological process.These factors form a network that means a complexity in psoriasis pathogenesis and treatment.Epigenetic modification is regarded as a vital mechanism in the pathogenesis of psoriasis.Histone methylation modifications is allowed for proper programming of the genome.Recent studies suggested that changes in histone methylation levels relying on histone modifier enzymes,may be related to psoriasis development,such as regulating production of some molecules.Lysine methyltransferase 2C(KMT2C),is a member of the type 2 lysine methyltransferase(KMT2A-D,F and G)family.KMT2C can bind at enhancers or promoters and regulate gene transcription.KMT2C is involved in the regulation of various biological functions,including cell development and senescence,DNA damage response,and sensitivity to endocrine hormone therapy.It has been shown that KMT2C is associated with some inflammatory diseases,such as rheumatoid arthritis.In psoriasis,the expression and biological role of KMT2C have not been studied,and the related regulatory mechanisms need to be further explored.Phosphoinositide-3-kinase regulatory subunit 3(PIK3R3),which encodes P55PIK(also called P55yγ,a part of the PI3K P85 regulatory subunit),is crucial for cell proliferation and downstream inflammatory signaling pathways,especially the PI3K/AKT pathway.A previous study showed that PIK3R3 could exacerbate psoriasis by facilitating IL-6 and IL-8 release and activating the AKT pathway.Long noncoding RNAs(lncRNAs)are emerging as key molecules in psoriasis.Gene expression can be regulated by lncRNAs through multiple mechanisms.This study was divided into three parts.Part 1 Expression in psoriasis and biological role in vitro of KMT2CObjective(1)To define the expression level of KMT2C in psoriatic lesion and cellular disease model.To study the effect of KMT2C on keratinocyte proliferation and psoriatic inflammation.(2)To predict and validate that the downstream target gene of KMT2C is PIK3R3,and molecular mechanism for regulating PIK3R3 expression.Methods(1)Thelesion tissues from patients with common psoriasis and skin tissues from healthy controls were collected,then the expression of KMT2C was detected using qRT-PCR and IHC.(2)Normal human epidermal keratinocytes and human immortalized keratinocytes(HaCaT and Ker-CT cell lines)were cultured and given M5 stimulation to construct a psoriasis-like cell model.The qRT-PCRand western blot were used to detect changes in the expression level of target gene PIK3R3 and the related signaling pathways.(3)CCK-8,EDU cell proliferation assay,flow cytometry cell cycle assay,western blot assay and ELISA assay were used to assess keratinocyte proliferation,cycle and psoriasis-related inflammatory factor expression levels after transfection of small interfering fragments of KMT2C.(4)We performed transcriptome RNA sequencing(RNA-seq)and analysis after KMT2C knockdown in M5-treated cells.The expression of the target gene PIK3R3 and changes and related signaling pathways were verified by qRT-PCR and western blot experiments.(5)The enrichment levels of H3K4me3 and H3K4me1 at the promoters and enhancers of PIK3R3 after KMT2C knockdown were determined using CUT&TAG experiments.Results(1)The expression of KMT2C was significantly upregulated in psoriatic lesion and was significantly higher in the M5-induced psoriatic keratinocyte model than controls in vitro.(2)KMT2C silencing resulted in that the keratinocyte proliferation were suppressed,cell cycle was arrested in the G1 phase,the expression levels of the cell cycle regulator cyclin D1 was reduced,low expression of IL-6,IL-8,CCL20 and S100A9 which upregulated by M5 stimulating.(3)The high level of PIK3R3 and activation of AKT/NF-κB signaling enhanced after M5 treatment were significantly reduced by KMT2C interference.(4)Silencing KMT2C diminished the enrichment of H3K4me1 and H3K4me3 at the PIK3R3 enhancer and promoter,which increased in M5-treated cells.Conclusion(1)The elevated expression of KMT2C promotes the keratinocyte proliferation and the secretion of psoriasis-associated inflammatory factors.(2)KMT2C may regulate PIK3R3 by targeting the PIK3R3 promoter and enhancer,then it can regulate the downstream AKT/NF-κB pathway.Part 2 The mechanism of the FABP5P3/KMT2C/PIK3R3 pathway in regulating cell proliferation and inflammationObjective(1)To investigate the role of PIK3R3 in KMT2C regulating psoriasis.(2)To test the expression level of FABP5P3 in skin lesion tissue of psoriasis patients and psoriasis-like cell model.(3)To verify whether FABP5P3 targeted KMT2C and the specific regulatory mechanism.Methods(1)After transfection of PIK3R3 plasmid(oePIK3R3)and cotransfection of siKMT2C and oePIK3R3,the effects of PIK3R3 on keratinocyte proliferation,cell cycle and psoriatic inflammatory responses were examined by CCK-8,cell cycle assay,western blot and ELISA assay.And the change in AKT/NF-κB signaling was examined by western blot experiments.(2)The expression levels of FABP5P3 in psoriatic lesions and psoriasis-like cell models were determined by immunofluorescence staining and qRT-PCR experiments.The levels of KMT2C after overexpression and knockdown of FABP5P3 were determined by qRT-PCR and western blot experiments.(3)The bioinformatics prediction website was used to search for RNA binding proteins that bound to both FABP5P3 and KMT2C.Immunofluorescence staining experiments was used to investigate the colocalization of FABP5P3 with RBP.RNA immunoprecipitation(RIP)assay were performed to verify the direct binding of RBP with FABP5P3 and KMT2C.(4)After FABP5P3 knockdown,RIP experiments were performed to verify the change in RBP binding to KMT2C,and the the degradation of KMT2C mRNA was revealed by actinomycin D mRNA stability assay.Results(1)Overexpressing PIK3R3 attenuated the inhibition of keratinocyte proliferation,downregulation of S100A9,IL-6,IL-8,and CCL20 and AKT/NF-κB signaling pathway that diminished by siKMT2C.(2)FABP5P3 expression is elevated in the skin lesion of psoriasis patients and psoriasis-like cell models.(3)The RNA binding protein HuR can bind to FABP5P3 and KMT2C.Knockdown FABP5P3 resulted in downregulation of KMT2C and reduced binding of HuR to KMT2C.Lack of FABP5P3 or HuR significantly accelerated KMT2C mRNA degradation.Conclusion(1)PIK3R3 may act as an essential mediator in KMT2C regulating AKT/NF-κB axis,which promotes proliferation and the M5-related inflammatory response in keratinocytes.(2)In psoriasis,upregulated FABP5P3 increases KMT2C mRNA stability by recruiting HuR.Part 3 KMT2C regulates epidermal proliferation and skin inflammatory response in psoriatic miceObjectiveTo clarify whether KMT2C is involved in the regulation of psoriatic epidermal hyperplasia and skin inflammation in vivo.Methods(1)Mice were used to induce psoriasis mice model by topical application of IMQ.The lesion severity was assessed by modified PASI score after mice were manipulated by intraepidermal injection of siKmt2c.The tissue of skin lesions was collected,and the histopathological sections were made,then HE staining was performed to verify the proliferation of epidermis.(2)The expression levels of KMT2C,PIK3R3 and proteins related to proliferation and inflammation were detected by qRT-PCR,western blot and IHC assay.(3)IHC technique was used to analyze the effects of KMT2C on the number of Ki-67+cells,and the chemotactic recruitment of CD3+T cells and CCR6+cells.Results(1)The mouse model of psoriasis was successfully induced by IMQ.Knockdown of KMT2C attenuates the degree of epidermal hyperplasia in psoriasis mice model.(2)Inhibition of KMT2C in the epidermis of psoriatic mice reduced the levels of PIK3R3 and inflammation-related proteins IL-6,IL-8,CCL 20,and S100A9 in skin lesions.(3)Knockdown of KMT2C reduced the infiltration of CD3+T cells and CCR6+cells in the skin of the IMQ-induced psoriatic mice.ConclusionDownregulation of KMT2C ameliorated the disease severity of psoriasis mice.