Functional Study Of E3 Ubiquitin Ligase RNF138 In Ulcerative Coliti

Ulcerative colitis(UC),which together with Krohn disease forms inflammatory bowel diseases(IBD)[1].Ulcerative colitis can be divided into active and remission stages,and its characteristic clinical symptom is bloody stool[4].UC may occur spontaneously,or due to changes in treatment or as a complication[4].The incidence rate of UC is the highest in late adolescence or early adulthood,and the incidence rate is equal in men and women[5].In recent decades,although the life expectancy of patients is improved upon the rapid development of medical treatment for UC,the incidence rate of UC has been growing rapidly worldwide,especially in developing countries.And the risk of recurrence after the disease is high,and there are still problems with current diagnosis and treatment.Therefore,research on UC diseases remains crucial.Recent studies have shown that E3 ubiquitin ligase plays a crucial role in the occurrence and development of inflammation.Ring Finger Protein 138(RNF138),also known as the NEMO like kinase related ring finger protein,has been characterized as an E3 ubiquitin ligase with multiple functional regions,including ubiquitin interaction motifs(UIM),RING domains,and C2HC and C2H2 zinc finger binding motifs[6-8].RNF138 was initially identified as interacting with NEMO-like kinases,leading to ubiquitination mediated TCF/LEF degradation,thereby negatively regulating of the Wnt signaling[9,10].RNF138 is proved to be involved in the regulation of secondary axis formation and the damage of colonic mucosal regeneration in the embryonic development of patients suffered from crohn’s disease[11],indicating that RNF138 plays a role in embryonic development,cell differentiation,cell proliferation,and cell regeneration.Interestingly,recent studies have shown that RNF138 can be recruited to regions of DNA double stranded breaks for participation in DNA damage repair systems through the regulation of homologous recombination[12].In addition,the downregulation of RNF138 is related to glioma cell apoptosis,indicating that RNF138 presents tumorigenic activityt[13].However,the molecular and physiological role of RNF138 in UC development is not well-established.The bioinformatics technology was utilized inthis study to integrate and analyze the microarray datasets,and detected the expression levels of RNF138 in different states of ulcerative colitis and colitis mucosa in the GEO database.We observed that the expression level of RNF138 in active UC patients was lower as compared to that in the normal controls.Upon immunosuppressive treatment,the expression level of RNF138 in active and noninflamed colon mucosa was significantly downregulated,indicating a significant decrease in RNF138 expression with the exacerbation of inflammatory levels in UC.Subsequently,clinical data also showed a negative correlation between the expression level of RNF138 and the severity of UC.To verify the above findings,RNF138 knockout and intestinal epithelial specific knockout mice were constructed and the UC models were achieved upon DSS feeding.In these two models,we observed hemorrhagic diarrhea and weight loss,as well as decreased colon length in mice after administration of DSS drugs.Within one week after the completion of the first administration cycle,significant weight loss and signs of disease were found,including hunchback,protruding fur,sepsis symptoms,and decreased mobility caused by diarrhea and anemia.Altogether,preliminary data in vivo experiments indicate that loss of RNF138 function can exacerbate DSS-induced ulcerative colitis.To further explore the mechanism accounting for the role of RNF138 in regulating the occurrence of inflammatory colitis,transcriptome analysis was employed on the intestinal tissues of the models mentioned above,as well as their respective control mice.We observed that RNF138 deregulation was correlated with the ectopic activation of NF-κB signal pathway in the intestine tissue samples as shown RNA-Seq.Finally,clinical tissue sample of UC was used to probe the correlation between RNF138 expression and the levels of p-p65,which indicated that the expression of RNF138 decreased and p-p65 staining increased in colon tissue of patients with UC.Further,a RNF138 knockout HCT116 cell line was constructed and employed to investigate the underlying mechanism of RNF138 in UC,indicating that the loss of RNF138 may be due to the upregulation of phosphorylation of NF-κB pathway proteins.Subsequently,the expression of downstream genes in the NF-κB pathway was detected and increased expression of PTGS2 was found.As a marker of ferroptosis,the expression level of PTGS2 significantly increased in cells with ferroptosis.We induced cell ferroptosis using RSL3 inducer and found an increase in ROS content and iron accumulation in the RNF138-KO cell population,indicating RNF138 deficiency-induced cell ferroptosis might be involved in the development of UC.However,the mechanism accounting for the UC exacerbation caused by loss of RNF138 function is still unclear,further investigation is needed.Overall,our data reveal that deregulation of RNF138 is correlated with UC development through negatively regulating the NF-κB signaling and thereby suppressing ferroptosis.