A Preliminary Study On Small Ubiquitination (SUMO)-modified Proteins In DSS Colitis And The Effect Of SUMO-modified Vitamin D Receptor On Its Regulatory Pathwa

Part I Effect of deSUMOylation on colitis and the regulation pathway of SUMOylation protein in colitisBackground:The incidence rate of ulcerative colitis(UC)has increased rapidly in recent years,and the pathogenesis is still unclear.Small ubiquitin-like modifier(SUMOylation)is an essential post-translational modification of proteins.Its modified proteins mediate various cellular functions such as transcriptional regulation and cell proliferation.SUMOylation of proteins is also the critical pathway for proteins in the pathogenesis of UC.However,the complete picture of proteins regulated by SUMO modification in the pathogenesis of UC is not completely clear.Objective:We used the sodium sulfate dextran(DSS)induced colitis model,to explore the pathways related to SUMOylation from the perspectives of proteomics and transcriptomics.It is expected that through this study,we can deeply understand the position of SUMOylation in the occurrence and development of UC and provide clues for finding therapeutic targets for UC in the future.Methods:1.5 mg/kg 2-D08 and/or DSS were used in mice to establish the deSUMOylation DSS colitis model.At the midpoint of the modelling,the mouse tail was cut to verify the expression of sumo.The colitis activity and pathological injury score were performed at the end of the modelling.The colonic tissue was taken for proteomic sequencing and analysis to verify the key proteins of the enrichment pathway.2.The DSS colitis transcript data were obtained from Gene Expression Omnibus(GEO).Moreover,it was analyzed by weighted gene co-expression network analysis(WGCNA)of small ubiquitin-like modifier(SUMO)E2-conjugating enzyme Ube2i related genes,the critical SUMOylation enzyme,and the key genes were verified.3.We integrated the key gene module and proteomic information and explored the SUMOylation related gene enrichment pathway.Results:1.(1)Compared with DSS colitis mice,the degree of colonic shortening and pathological injury in deSUMOylation DSS colitis mice was minor(P-value<0.05).(2)Proteomic analysis showed multiple pathways affected in the colon tissue of deSUMOylation colitis,involving the protective and pro-inflammatory pathways of inflammation.The deSUMOylation colitis group activated inflammatory protective pathways such as Rap1-p38mapk signalling pathway and adhesion junction pathway and affected inflammatory pathways such as neutrophil extracellular traps(NETs)formation pathway and bile acid metabolism pathway.(3)Compared with DSS colitis,the key pathway proteins,NE was no change in the expression(P-value=0.313),myeloperoxidase(MPO)(P-value=0.0244)and p38MAPK were decreased(P-value=0.043)in the colon tissue of deSUMOylation colitis.2.(1)DSS colitis data sets GSE42768,GSE134281 and GSE71920 were screened from the GEO database.A total of 5464 Ube2i related genes were screened from the combined data set after removing the batch effect;There are 286 differentially expressed genes(DEGs)which can be used for subsequent WGCNA.(2)The brown gene module(correlation coefficient=0.87)and turquoise gene module(correlation coefficient=-0.83)obtained by WGCNA strongly correlated with colitis.The brown gene module was enriched in protein synthesis and processing pathways,while the turquoise module was enriched in infection,metabolism,and drug response pathways.(3)The relative mRNA expressions of hub genes Sulf2,Rar,Cyp2f2,Esrrg,Ube2i and Vdr,were verified.The results showed that compared with the control group,the expression of Sulf2,Rar,and Ube2i increased,and the expression of Cyp2f2,Esrrg and decreased in the colon of colitis mice.3.We integrated proteomic and transcriptional information,and the results showed that Ube2i was involved in trans nuclear membrane transport and NF-κB signalling pathway,ubiquitin-mediated protein degradation pathway.The SUMOylation related protein VDR participates in the calcium reabsorption pathway and receptor activation pathway and regulates gene transcription.Conclusions:1.Treatment of the DSS colitis model with a UBE2I inhibitor(2-D08)can affect DSS inflammatory response.2.Proteomic and transcriptomic analysis showed that SUMOylation modified crucial proteins of multiple signal pathways in colitis.Part II SUMOylation of vitamin D receptor in effecting its protein stability and the function of colonic epithelial cellsBackground:Vitamin D receptor(VDR)regulates many functions,such as cell proliferation,apoptosis,inflammatory response,etc.Previous studies have shown that VDR is a sumo modified protein,but little is known about the effect of SUMO modification on the downstream pathway of VDR and the effect of SUMO modification regulating VDR on colonic epithelial function.Objective:To clarify the correlation and interaction between VDR and SUMOylation,construct the SUMOylation mutant of VDR,and explore the effect of SUMOylation on the protein stability of VDR and the function of colonic epithelial cells.Methods:1.Correlation between VDR and SUMOylation:analyze the expression of VDR and SUMO in the public data set GSE16879,collect five UC patients and five non-UC patients undergoing colonoscopy,and explore the mRNA expression and correlation of VDR and SUMO in colon tissue by qPCR experiment.2.Interaction between VDR and SUMO:immunoprecipitation was carried out with mouse colon tissue.3.Construction of VDR SUMOylation mutation cells:carry out SUMOylation sites mutation on VDR;construct VDR SUMOylation mutant plasmid;package virus with VDR SUMOylation mutant plasmid and VDR plasmid,and construct stable expression transfection cell line.4.Effect of SUMOylation on VDR:the effect of cycloheximide(CHX)on the protein stability of VDR was detected by VDR SUMOylation mutant cells and VDR overexpressed cells.5.SUMOylation and VDR regulated functions:MG132 intervention cell lines to explore the ubiquitination and degradation pathway of VDR.Cell proliferation assay(CCK-8)was used to detect the effect of SUMOylation of VDR in cell proliferation under TNF-α(and vitamin D analogues)intervention.We used TNF-α to investigate the effect of SUMOylation in VDR on the expression of cell tight junction proteins and inflammatory factor IL-1β.Results:1.Compared with non-UC patients,the expression of VDR and SUMO protein decreased in UC patients in the public dataset(P-value<0.001),while in our validation results,the expression of VDR in UC patients decreased(P-value=0.0127),and the expression of SUMO2/3 tend to decrease.There was a positive correlation between SUMO2/3 and VDR(correlation coefficient=0.49).2.The mice’s colonic tissue was immunoprecipitated with VDR and then immunoblotted with SUMO2/3 antibody.The results showed a migration band at the molecular weight of VDR,and there was an interaction between VDR and SUMO2/3;that is,VDR was a SUMOylation protein.3.We mutated the lysine(AAG)at sites 91,103 and 111 of the N-terminal of VDR into arginine(AGG).Then,the Flag tag sequence was added to the 5 ’end of VDR,and the SUMOylation mutated VDR and connected the sequence to the lentivirus vector pLV-EF1a-EGFP(2A)Puro.We used the packaged virus to infect HCT116 cells.After screening,VDR and SUMOylation mutant cells(3K3R cells)of VDR were obtained.The expression of enhanced green fluorescent protein(EGFP)was expressed in obtained cell lines.4.After CHX intervention in different time points,the protein level of VDR gradually decreased in all obtained cell lines,while it decreased more significantly in 3K3R cells.5.(1)After MG132 intervened at different time points,the VDR protein level increased in the VDR cells while relatively stable in the SUMOylation mutant cells.(2)Intervened by TNF-α,the proliferation rate of cells in the SUMOylation mutant cells was higher than that in VDR cells(final cell proliferation multiple:3K3R vs VDR 1.02 ± 0.06 vs 0.90±0.07;P-value=0.0002).However,the proliferation rate in 3K3R and VDR cells was lower than that of control cells.After pretreatment with vitamin D analogue calcipotrio,the proliferation efficiency of VDR or 3K3R cells was lower than that of the corresponding cells without calcipotrio treatment(P-value<0.0001),while the difference between VDR and 3K3R cells overtime was also reduced(P-value=0.02).(3)The experimental results of qPCR and Western blot suggest that after TNF-αtreatment,the mRNA and protein levels of E-cadherin,occludin and ZO-1 in epithelial cells were decreased,while the expression of tight junction proteins in VDR cells was higher than that in control and 3K3R cells.(4)After 12 hours of TNF-α stimulation,the expression of IL-1β was significantly lower in VDR cells than in the control cells,but there was no difference with 3K3R cells.Conclusions:1.there is a correlation between VDR and SUMO protein expression,and VDR is a SUMOylation protein.2.There are three SUMOylation sites in VDR.After mutation of all SUMOylation sites,the degradation rate of VDR protein was increased through the ubiquitination pathway.Sumoylation can maintain the anti-proliferation of VDR in epithelial cells,and maintain the stability of VDR mediated tight junction function.Part III VDR is an important protein for predicting the treatment of ulcerative colitis with TNF-α inhibitorsBackground:Infliximab is the most widely used biological agent in treating patients with ulcerative colitis(UC).However,one-third of UC patients have primary non-response(PNR).There is no effective index for predicting PNR in the clinic.Objectives:To screen the signatures that can be used to predict the primary IFX non-respond in patients with UC from the transcriptome database and explore their stability and feasibility of an application.Methods:1.Three GEO data sets were analyzed,and the Robustrank Aggreg(RRA)algorithm was used to screen the integrated differential expression genes between IFX response and non-response to construct the prediction model.2.The bootstrap analysis method that,combined with resampling and artificial neural network(ANN)analysis,was used to evaluate the predictive value of the model,and the independent transcriptome data set was used to verify it.3.A clinical immunohistochemical(IHC)experiment was used to explore the expression of the indicators in the model at the protein level,and ROC curve analysis was used to screen the prediction threshold.Results:1.We used the RRA algorithm to screen out six gene combinations(CDX2、CHP2、HSD11B2、RANK、NOX4 and VDR)to predict the primary failure of IFX treatment in patients with UC.2.The bootstrap method showed that the predictive value of the model composed of six genes for the primary loss of response to IFX was 0.850±0.103.The AUC range of the independent validation data set was 0.759±0.065.The prediction results are stable.3.After the IHC experiment,the IHC staining results of VDR and RANK were significantly correlated with the efficacy of IFX(P-value<0.05).In pretreatment colonic tissue,the total IHC score of VDR less than five and RANK less than 7 has an AUC of 0.828(95%CI:0.665～0.991,P-value=0.013)in predicting PNR,the sensitivity was 82.4%,and the specificity was 71.4%.Conclusions:1.The result showed a connection between the RNA and protein model,and both two models can be used in predicting the primary failure of IFX.2.The protein model of VDR combined with rank has a more robust clinical application.