OTUB1 Deubiquitinates TGFBI Through Non-canonical Pathway And Mediates The Progression Of Infantile Hemangioma By Regulating Glycolysis

Background and purpose:Infantile hemangioma(IH)is one of the most common vascular tumors in infants with an incidence rate of 4.5%.Female gender is the risk factor for developing IH.IH patients without prompt treatment often leave varying degrees of secondary deformities.And 10%of the patients suffer serious complications including hemorrhage,skin necrosis,and Airway obstruction because of the fast proliferation and special location of IH,which are life-threatening and usually induce great damage in appearance as well as function.At present,it is still unclear regarding the biological mechanisms underlying the development of IH.Ubiquitin system controls the degradation,stability and functional status of protein by ubiquitination,which regulates many physiological and pathological processes.A great deal of evidence indicated that deubiquitylase OTUB1 plays an important role in multiple cancers.The function of OTUB1 in IH is still blank.The purpose of this study is to investigate the function of OTUB1 in IH progression,find new therapeutic target and improve the therapeutic effect for IH.Methods:1.The expression level of all the deubiquitylases in IH tissues was detected by transcriptome sequencing.The results were analyzed and OTUB1 was predicted to play an essential role in IH as deubiquitylase.2.OTUB1 knockdown(or expression)lentivirus was constructed.Hemangioma endothelial cells(HemECs)with stable overexpression or knockdown of OTUB1 were constructed using the lentivirus and HemECs isolated from IH tissues.CCK8assays,transwell assays and tube formation assays were performed to investigated the function of OTUB1 in HemECs in vitro.Nude muse model for IH was used to assess the role of OTUB1 in the progression of IH in vivo.3.Mass spectrometric analysis and transcriptome sequencing were performed to investigate the function OTUB1 in IH and TGFBI was predict to be the downstream substrate protein of OTUB1.Co-IP assays,pulldown assays,and immunofluorescence co-localization,ubiquitination assays and half-time assays were performed to verified the ubiquitination sites of TGFBI as well as the interaction between OTUB1 and TGFBI.4.OTUB1 missense mutants and TGFBI mutant on ubiquitination sites were constructed to investigate the further regulation mechanism between OTUB1 and TGFBI.5.Rescue experiments were performed to evaluated whether TGFBI was a critical functional target of OTUB1 in IH.Given that TGFBI is associated with glycolysis,the metabolic condition of HemECs and the expression level of glycolysis related proteins were measured to further investigate the mechanism that OTUB1 promotes IH progression by deubiquitinating TGFBI and regulating glycolysis.Results:1.The results of transcriptome sequencing indicates that OTUB1 is the deubiquitylase with highest expression level in IH.2.In vitro experiments suggested that OTUB1 knockdown inhibits the proliferation,migration and tube formation ability of HemECs and OTUB1 overexpression promoted these abilities.In IH mouse models,the His with OTUB1 knockdown showed lighter color,lower density of micro vessels and less expression of angiogenesis related molecules like VEGFA.3.The results of proteomic analysis indicated that TGFBI is a possible functional downstream protein deubiquitinated by OTUB1.Immunofluorescence showed that OTUB1 and TGFBI colocalized in cytoplasm.Co-IP and pulldown assays proved the directly interaction between OTUB1 and TGFBI.OTUB1 could decrease the ubiquitination level of TGFBI and increase the stability of TGFBI.The ubiquitination sites of TGFBI are on K22 and K25.4.OTUB1 missense mutants,which lost the enzymatic activity could still deubiquitinated TGFBI.The ubiquitination level of TGFBI mutant on ubiquitination sites was significantly decreased.5.In the rescue experiments,TGFBI overexpression could enhance the proliferation,migration and tube formation ability of HemECs in vitro,even though OTUB1 knockdown inhibits this trend.In vivo,TGFBI overexpression reversed the inhibition of IH progression induced by OTUB1 knockdown.In addition,The knockdown of OTUB1 could reduce the expression of glycolysis related molecules such as HIF-la,GLUT-1 and HK-2 and downregulate the glycolysis level in HemECs by decreasing TGFBI expression.Conclusion:OTUB1,which is highly expressed in IH tissues,could promote the proliferation,migration and tube formation ability of HemECs in vitro and accelerate IH progression in vivo.OTUB1 plays its role in IH by deubiquitinating its downstream substrates protein TGFBI.OTUB1 could directly interact with TGFBI,deubiquitinate TGFBI and increase its stability,which is independent of its enzymatic activity.OTUB1-TGFBI axis was an important pathway by which OTUB1 regulated progression and angiogenesis of IH by mediating glycolysis.This finding not only provided new insights into the role of OTUB1 in glycolysis and angiogenesis,but also clarified the mechanism of IH pr0ogression from the perspective of ubiquitination,found new effective therapeutic target and provided novel therapeutic strategies.