Role Of NDRG2 In Initiation And Progression Of Colitis And Colitis Associated Colorectal Cancer

Inflammatory bowel disease(IBD)is a multifactorial chronic inflammatory disease mainly comprising Crohn’s disease(CD)and ulcerative colitis(UC)that causes intestinal epithelial cell injury and relapsing chronic pathogenic rectal and colonic inflammation.IBD emerges as an important kind of disease harming the patient health.Remarkably,chronic inflammation induces the colitis associated colon cacer(CAC).The association between colitis and C AC is well estabilished,even though the genetic changes corresponds to the initiation of CAC,the inflammation during colits promotes the progression via the enhancement of IL-1,IL-6,TNF-α and other factors.The paghogenesis of colitis correlated with the food,environmental factors and immune homeostasis,which accelerates the formation of CAC.Elucidating the paghogenesis of colitis assists the therapy and diagnosis of CAC.N-myc down-stream regulated gene(NDRG2)was firstly found and characterized in golima cells.As a tumor suppressor gene,NDRG2 inhibited proliferation,invasion,metastasis of tumor cells,and promoted apoptosis,repair of DNA damage and sensitivity to chemotherapeutics.Especially in colon cancer,we also demonstrated that NDRG2 could act as a novel prognosis marker.However,the function of NDRG2 is still needed to be further demonstrated.Otherwise,whether NDRG2 participated the pathogenesis of colitis is still il usive.To further understand the function of NDRG2,we constructed intestine-specific conditional Ndrg2 knockout mice(Ndrg2flox/flox)using Cre/Loxp system,a nd we induced colitis and colitis associated colon cancer via DSS or AOM/DSS treatment to elucidate the function of NDRG2 in paghogenesis of colitis and CAC,thus we further investigated the role of NDRG2 in tumorigenesis of colon cancer.【Objectives】 1.To investigate whether NDRG2 involved in initiation and progression of colitis.2.To investigate the role of NDRG2 in pathogenesis of colitis.3.To explore the role of NDRG2 in the transformation from colitis to colitis associated colon cancer(CAC).【Methods】 1.Expression of NDRG2 decreased in DSS and LPS induced colitis.We firstly investigated that DSS induced obvious phenotype of colitis,however,there’s evident difference between male and female mice,and it seemed that the male mice exhibited a higher sensitivity to DSS-induced colitis than female.During the progression of colitis,NDRG2 decreased obviously in intestinal epithelial cells(IECs).Moreover,through the injection,expression of NDRG2 in IECs was reduced identically.Thus,N DRG2 was decreased during the colitis progression,suggesting the functional role of NDRG2 in colitis.2.Deletion of NDRG2 increased susceptibility to DSS-induced colitis.We constructed intestine-specific conditional Ndrg2 knockout mice(Ndrg2flox/flox,ΔIEC)and verified that expression of NDRG2 was totally loss in IECs.WT and ΔIEC mice were treated with DSS,and were assigned as WT-DSS and ΔIEC-DSS.ΔIEC-DSS mice exhibited increased susceptibility to DSS-induced colitis,with increased loss of body weight,loss of colon length,epitheliaum destruction and decreased survival rate.Furthermore,absence of NDRG2 enhanced the recruitment of monocytes,macrophage and neutrophils via the up-regulation of proinflammatory chemokines like C XCL-1,CXCL-2 and CCL-5 in IECs from ΔIEC-DSS mice,and we also demonstrated the expression of NDRG2 was negatively correlated with recruitment of CD68 positive macrophage in human UC samples.Deletion of NDRG2 also induced the expression of proinflammatory cytokines after treatment of DSS,like IL-1β,IL-6 and TNF-α,and suppressed the expression of IL-10,an anti-inflammatory cytokine,thus accelerated the progression of colitis.During the paghogenesis of colitis,the phosphorylation of STAT3 exhibited no difference between WT-DSS and ΔIEC-DSS,but ΔIEC-DSS mice showed an increased activity of NF-κB,possibly lead to the progression of colitis.Taken togegher,deletion of NDRG2 increased susceptibility to DSS-induced colitis.3.Absence of NDRG2 disrupted the adherens junction(AJ)within colonic epithelial cel s.Dysfunction of junctional structure contributed to the increased permeability of colon epithelium,thus induced initiation and progression of colitis.To further investigate the role of NDRG2 in colitis development,we treated the WT and ΔIEC mice with FITC-Dextran and LPS,and we found that deletion of NDRG2 increased the permeability of colonic epithelium,for the increased concentration of serum FITF-Dextran and serum LPS in ΔIEC mice.Because that the increase of permeability was corresponding to the destruction of junctional structure,we detected the structure of colonic epithelium between WT and ΔIEC mice,and the adherens junction was obviously abnormal in ΔIEC mice.We hypothesized that the destruction of adherens junction was dut to the suppression of E-cadherin in colonic epithelium from ΔIEC mice.Further analysis showed that the expression of E-cadherin was evidently inhibited at mRNA and protein level,possibly because the increased expression of Snail and Slug.This evidence was also verified in HT-29 and Caco-2 cell lines via the inhibition of NDRG2 using siRNA.In human colonic epithelial cells,the expression of NDRG2 was positively correlated with E-cadherin,thus supported our hypothesis that absence of N DRG2 lead to the inhibition of E-cadherin via up-regulation of Snail and Slug,resulting in the abnormity of adherens junction,thus increased the susceptibility to colitis initiation and progression.4.Deletion of NDRG2 induced the initiation and progressio n of colitis associated colon cancer(CAC).AOM-DSS system was much efficient to induce the colitis associated colon cancer.AOM could induce the mutation of β-catenin and suppressed its degradation,leading to the initiation of colon cancer,together with the colitis progression induced by DSS,AOM-DSS treatment finally induced the tumorigenesis of colitis associated colon cancer(CAC).After the treatment of AOM-DSS,we found ΔIEC mice exhibited increased numbers and malignance of tumors,compared with the WT group,thus demonstrated that deletion of NDRG2 facilitated the initiation and progression in colitis associated colon cancer(CAC).【Conclusion】In our study,we found that NDRG2 regulated the pathogenesis of colitis for the first time.Dysregulatio n of NDRG2 inhibited the expression of E-cadherin and disrupted the adherens junction within colonic epithelium,thus increased susceptibility to colitis pathogenesis.Moreover,we also found that deletion of NDRG2 induced the initiation and progression of colitis associated colon cancer(C AC).Taken together,we demonstrated the role of NDRG2 in initiation and progression of colitis and colitis associated colon cancer(CAC).Based on our study,NDRG2 could be regarded as a novel marker for the diagnosis and therapy of colitis and colitis associated colon cancer(CAC).