The Role Of CircEIF5 In Regulating Keratinocyte Proliferation And Inflammation In Psoriasis

Research BackgroundPsoriasis is a chronic,relapsing,immune-mediated systemic inflammatory disease characterized by scaly,well-defined erythema,with hyperproliferation and abnormal differentiation of keratinocytes as its main hallmarks[1].It is characterized by high incidence,easy recurrence,easy drug resistance and multiple comorbidities,which seriously reduces the quality of life of patients.The pathogenesis of psoriasis involves a variety of internal and external factors,including immune abnormalities,genetic factors,and even environmental factors,but the specific mechanism is still unclear.In recent years,the development of epigenetics has advanced by leaps and bounds.More and more new mechanisms regulating diseases such as non-coding RNA,DNA methylation,histone modification and chromatin remodeling have been discovered.A variety of microRNAs（miRNAs）and long non-coding RNAs（lncRNAs）are involved in the regulation of keratinocyte proliferation and inflammatory cell activation in psoriasis,but the research on circular RNAs（circRNAs）in psoriasis is still relatively limited.However,its mechanism of regulating the pathogenesis of psoriasis remains to be further explored.Circular RNAs（circRNAs）are a class of endogenous non-coding RNAs produced by exon skipping or back-splicing without 5’-3’ polarity and polyadenylated tails.CircRNAs are highly evolutionarily conserved and stable,and are cell-and tissue-specific[2].Accumulating evidence indicates that many circular RNAs have important biological functions and are involved in the initiation and progression of various diseases,including cancer,autoimmune diseases,and metabolic diseases.In psoriasis,some circRNAs,such as hsa_circ₀003738 and hsa circ₀061012,have been reported to regulate keratinocyte proliferation,migration,and invasion and participate in the dysfunction of regulatory T cells（Tregs）,but most of the studies focused on cirRNAs Described as a sponge of microRNA（miRNA）,it works by competing with miRNA for binding to miRNA,and current research has found that cirRNA can also regulate biological effects by regulating proteins,self-encoded polypeptides,etc.The way in which cirRNA acts through regulatory proteins in disease is still blank.High mobility histone B1（high mobility group box-1,HMGB1）is a late inflammatory factor,usually involved in the maintenance of genome stability and regulation of gene transcription[3],but can be actively or passively released after stress In the extracellular fluid,it can bind to ligand advanced glycation end products（RAGE）and toll-like receptors（TLRs）,and participate in the regulation of NF-κB signaling pathway to cause cell proliferation,apoptosis and inflammatory response[4].In psoriasis,HMGB1 has been widely confirmed to promote the activation of keratinocyte NF-κB signaling pathway and inflammasome by altering subcellular localization and autocrine,and promote various related inflammatory factors such as CXCL1,CXCL2 and CCL20.increased secretion[5].Our research group used the cirRNA expression profiling chip technology to analyze the differential cirRNA in psoriasis tissue[6],and found that the expression of circEIF5 was significantly up-regulated in psoriasis vulgaris skin lesions.The regulation of keratinocyte proliferation and inflammation in psoriasis and the related signaling pathways it may be involved in were investigated.And by exploring its downstream binding proteins to reveal the mechanism of circRNAEIF5 in the pathogenesis of psoriasis.Objectives:1.To clarify the expression of circEIF5 in psoriatic skin lesions and in vitro cell models of psoriasis,and to verify its ring structure.2.To study the effect of circEIF5 on keratinocyte proliferation and pro-inflammatory chemokine secretion and its possible related signaling pathways.3.Explore the involvement of circEIF5 in keratinocyte chemokine secretion by regulating HMGB1 protein.Part 1 Analysis of the expression level of circEIF5 in psoriatic skin lesions and psoriasis cell models and confirmation of its ring structureMethods:（1）qRT-PCR was used to detect the expression of circEIF5 in skin lesions of patients with psoriasis vulgaris and skin tissues of healthy controls.（2）qRT-PCR was used to detect the expression level of circEIF5 in a psoriasis cell model constructed from human immortalized keratinocytes（HaCaT cells line）pretreated with M5.Nuclear separation of HaCaT cells were carried out and the expression of circEIF5 in the nucleus and cytoplasm was detected by qRT-qPCR.（3）The circular structure of the circEIF5 sequence was verified by Sanger sequencing,RNase R enzyme digestion and forward and reverse PCR.Result:（1）qRT-PCR detection showed that the expression of circEIF5 in psoriasis vulgaris skin lesions was generally higher than that in normal skin tissue.（2）qRT-qPCR results of in vitro cell experiments showed that the expression level of circEIF5 in the keratinocyte psoriasis model pretreated with M5 was significantly up-regulated compared with the control group.CircEIF5 was expressed in both the cytoplasm and the nucleus,but mainly in the cytoplasm.（3）Sanger sequencing,RNase R enzyme digestion and forward and reverse PCR all confirmed that circEIF5 is a circular structure.Part 2:CircEIF5 regulates keratinocyte proliferation,chemokine secretion and related mechanisms by activating NF-kB and STAT3 signaling pathwaysMethods:（1）siRNA was transfected in M5-stimulated HaCaT cell psoriasis model to interfere the expression of circEIF5;circEIF5 overexpression plasmid was constructed and transfected into HaCat cells not stimulated by M5.CCK-8 and flow cytometry were used to detect the effect of interference and overexpression of circEIF5 on cell proliferation and cell cycle.The effects of overexpression and interference of circEIF5 on the expression levels of cell cycle-related proteins were detected by Western blot assay.ELISA was used to measure the secretion levels of chemokine（CXCL2,CXCL8,CXCL10,CCL20）in M5-stimulated HaCaT cells.（2）Western blot was used to detect the expression of key protein molecules of NF-κB and STAT3 signaling pathway in HaCaT cells transfected with overexpression circEIF5（OE-circEIF5）plasmid and M5-stimulated HaCaT cells transfected with siRNA.（3）HaCaT cells transfected with circEIF5 overexpression plasmid were intervened with NF-κB and STAT3 pathway inhibitors（JSH-23 and stattic）,the cell proliferation and cell cycle were detected by CCK-8 and flow cytometry.Westenblot assay were used to measure the expression levels of cell cycle-related proteins.The effects on the secretion levels of chemokines（CXCL2,CXCL8,CXCL10,CCL20）were detected by ELISA.Result:（1）The siRNA of circEIF5 was transfected in the M5-induced HaCaT cell psoriasis model,and the qRT-PCR results showed that the expression level of circEIF5 was significantly down-regulated;the results of the CCK-8 cell proliferation experiment showed that the M5-induced cell proliferation was significantly inhibited;The results of the cycle experiments showed that the shortening of the G1 phase and the prolongation of the S phase of the cell cycle induced by M5 were also inhibited;Western blot experiments showed that the expressions of cell cycle-related proteins CyclinD1 and C-Myc were decreased.（2）HaCaT cells were transfected with circEIF5 overexpression plasmid.qRT-PCR results showed that the expression level of circEIF5 was significantly increased.The results of CCK-8 cell proliferation experiments showed that HaCaT cells proliferated actively.The results of cell cycle experiments showed that the G1 phase was shortened and the S phase was prolonged;Western blot experiments showed that the expressions of cell cycle-related proteins CyclinD1 and C-Myc were increased.（3）Interfering with the.expression of circEIF5 in HaCaT cells could inhibit M5-induced chemokine secretion.While overexpression of circEIF5 in HaCaT cells could promote M5-induced chemokine secretion.（4）Disruption of circEIF5 in M5-induced HaCaT cell psoriasis model reduced the expression of p-p65 and p-STAT3.In contrast,overexpression of circEIF5 in HaCaT cells increased the levels of p-p65 and p-STAT3.（5）Both JSH-23 and stattic could inhibited the proliferation of HaCaT cells and the shortening of the G1 phase of the cell cycle induced by overexpression of circEIF5.Stattic can inhibit the overexpression of circEIF5-induced increase in the expression of cycle-related proteins CyclinD1 and C-Myc in HaCaT cells,but JSH-23 can only inhibit the high expression of CyclinD1.（6）Both JSH-23 and stattic could inhibite the chemokine secretion in HaCaT cells induced by OE-circEIF5.Part 3:CircEIF5 regulates subcellular localization and secretion of HMGB1 proteinMethod:（1）Construct the overexpression plasmid of circEIF5 sequence and its complementary sequence for in vitro transcription.After in vitro transcription,use RNA pull-down experiment to enrich the protein interacting with circEIF5 sequence.SDS-PAGE electrophoresis silver staining detects the enriched protein.Chromatographic mass spectrometry analysis identified proteins that may interact with CircEIF5.Western blot was used to further verify that HMGB1 protein was enriched in circEIF5 sequence RNA pull-down experiment products.RN A protein binding immunoprecipitation（RIP）was used to enrich the RNA bound to HMGB1 protein,and RT-PCR was used to verify the presence of circEIF5 in the product.（2）The content of HMGB1 protein in the cell culture supernatant after knockdown of circEIF5 in M5-stimulated HaCaT cells was detected by ELISA.（3）The expression of HMGB1 protein in the nucleus and cytoplasm of M5-stimulated HaCaT cells after knockdown of circEIF5 was detected by westen blot method.Result:（1）Both RNA pull-down experiments and RNA-protein binding immunoprecipitation（RIP）confirmed that circEIF5 binds to HMGB1.（2）ELISA showed that knockdown of circEIF5 in M5-stimulated HaCaT cells reduced M5-induced HMGB1 extracellular secretion.（3）Westenblot’s method confirmed that when circEIF5 was knocked down in M5-stimulated HaCaT cells,the protein content of HMGB1 was increased in the nucleus,while the protein content of HMGB1 in the cytoplasm was decreased.Conclusion:（1）The expression of circEIF5 was increased in both psoriatic lesions and M5-stimulated keratinocytes,and it was indeed a circular structure.（2）CircEIF5 is involved in the regulation of HaCaT cell proliferation and chemokine secretion by regulating NF-κB and STAT3 signaling pathways.（3）CircEIF5 participates in regulating the occurrence and development of psoriasis by targeting the regulatory protein HMGB1.