| Background and ObjectiveIntestinal mucosal barrier is composed of mucus and epithelial cells.Mucin was synthesized or secreted by epithelial cells,which is mainly composed of mucin-type O-glycans.Therefore,the function of mucin is related with the O-glycans,recent studies have indicated that intestinal mucin O-glycans severved as some bacterial adhesion sites.The attachment of cell-surface O-glycans to proteins are mediated by serine,threonine and proline and involved in tumor invasion and metastasis,and patient prognosis.However,host glycosylation in interactions with EPEC and EHEC O157:H7,and how O-glycans trigger signaling to control tumor metastasis is largely unknown.Mucin-type O-glycans are initiated by the GALNT(UDP-GalNAc: polypeptide GalNAc transferase)family of glycosyltransferases whose members transfer N-acetylgalactosamine(GalNAc)to serine or threonine residues on apomucins to form a Tn epitope.O-glycans are sequentially extended by a series of enzymatic reactions that are classified into eight major groups(Cores 1–8)based on the addition of different carbohydrate residues to GalNAc-Ser/Thr.Therefore,we inhibited the synthesis of O-glycans by benzyl-α-GalNAc to investigate whether O-glycans were involved in E.coli adherence and invasion into intestinal epithelial cells,we inhibited Core 2 O-glycan synthesis by sh RNA targeting C2 Gn T2 to investigatei its role in bacteria adherence or invasion into intestinal epithelial cells.We overexpressed Core 3 O-glycans synthase(β3gn T6)by lentivirus to explore its effect on the molecular mechanism of mucin-type Core 3 O-glycan in modulating EMT-MET plasticity of CRC cells.The renewal and differentiation of intestinal epithelial cells is the critical mechanism for regulating intestinal epithelial mucosal barrier.Ascl2 regulated intestinal crypt stem cells are important source of turnover in intestinal epithelial mucosa.Recent studies have found that Ascl2 is able to transactivate its own promoter in intestinal cryptic Lgr5+ stem cell,together with β-catenin/Tcf,activates the genes fundamental to the stem cell state.This result encourages us to infer whether Ascl2 autoregulation is present in colon cancer progenitor cells and its biological significance.Therefore,the CD133+CD44+ and CD133-CD44-cell population from HT-29 or Caco-2 cells were isolated of by fluorescence-activated cell sorting and flow cytometry,and the cells were treated in their different culture media contained 5,10,20,40,80,200,500 or 1000 ng/ml Wnt /R-spondin1 for 16 hours to explore its effect on molecular mechanism of Ascl2 autoregulation.Methods1.Mucin-type O-glycans in regulating bacteria adherence to and invasion into intestinal epithelial cells1.1 Culture of HT-29 cells in glucose-free,galactose-substituted cell culture medium for 7 days with a medium change each day caused HT-29 cell differentiation,and these differentiated cells are referred to as HT-29-Gal cells.1.2 The intestinal epithelial cells(HT-29 and HT-29-Gal)were treated with benzyl-α-GalNAc(O-glycans synthesis inhibitor)for 7d,and the O-glycan-deficient cells were named as HT-29-OBN and HT-29-Gal-OBN cells.1.3 Lectin histochemical staining(ConA,GSL-II,MAA,PNA,DBA,UEA-I)was performed in HT-29,HT-29-OBN,HT-29-Gal or HT-29-Gal-OBN cells to confirm their glycosylation alteration.1.4 Bacterial adherence and invasion to the relative cells was determined using the direct co-staining of adherent bacteria and host cells,the adherent bacteria plating,and/or the direct fluorescent observation of the adherent GFP-labeled bacteria.1.5 To investigate the role of Core 2 O-glycans in bacteria(EPEC or EHEC O157: H7)adherence or invasion into intestinal epithelial cells which were inhibited for Core 2 O-glycans via sh RNA targeting C2GnT2.1.6 The shRNA-Ctr/HT-29 and sh RNA-C2GnT-2/HT-29 cells infected with bacteria or not were measured by TEER and stained for tight junction protein(ZO-1 and occuludin)by immunohistochemistry to observe their epithelial barrier function.2.Mucin-type Core 3 O-glycans in regulating the epithelial-mesenchymal transition(EMT)-mesenchymal-epithelial transition(MET)plascity in colon cancer cells2.1 Samples from normal mucosa and cancer tissues were investigated the corelation between Core 3 synthase expression status and the clinical characteristics of the CRC patients(lymph nodes metastasis,distant metastasis and prognosis)by analyzing the TCGA cohort.2.2 HT-29 and LS174 T cells were transfected with lentivirus particles using a LV5(EF-1aF/GFP&Puro)vector with a Core 3 synthase insert(β3gnT6/HT-29 or β3gnT6/LS174T).The MTT,migration,invasion and the ability of vivo tumorigenicity experiments were used to investigate the effect of Core 3 synthase re-expression in colon cancer cells on cell biological behavior.And PCR and Western blot experiments were used to detect the change in EMT-related markers(E-cadherin,N-cadherin,snail,slug,ZEB1 and ZEB2).2.3 Lectin histochemical staining,SDS-agarose electrophoresis and Coomassie Brilliant Blue staining were used to detect the change of O-glycan structures in Core 3 synthase re-expression cells.PCR and/or Western blot experiments were used to detect the expression of miR-200 c or p53.2.4 Transfection of luciferase reporters containing p53 or mi R-200 c promoter into Ctr/HT-29 and β3gnT6/HT-29,Ctr/ LS174 T and β3gn T6/LS174 T cells was used to confirm wether Core 3 O-glycan restoration led to activation of p53 or mi R-200 c promoters.MUC1-C binding to the p53 proximal promoter and p53 binding to the mi R-200 c proximal promoter were proven by ChIP assays.2.5 EMT-MET plasticity in mucin-type Core 3 O-glycan restorated CRC cells was further clarified by inhibit of MUC1-C nucleus translocation(GO201),MUC1 si RNA or p53 si RNA,mi R-200 c inhibitor.3.Ascl2 autoregulation in CRC cells3.1 PCR and Western blot experiments were used to determine whether exogenous Ascl2 in colon cancer cells activated the endogenous Ascl2 expression.3.2 The exogenous Ascl2 in colon cancer cells activated the endogenous Ascl2 expression via a direct autoactivatory loop,including Ascl2 binding with its own promoter and transcriptional activation of Ascl2 gene were further clarified by Ch IP and luciferase experiments.3.3 The CD133+CD44+ and CD133-CD44-cells population from HT-29 or Caco-2 cells were isolated of by fluorescence-activated cell sorting and flow cytometry.3.4 The CD133+CD44+ and CD133-CD44-cells were treated in their different culture media contained 5,10,20,40,80,200,500 or 1000 ng/ml Wnt/R-spondin1 for 16 hours,PCR and Western blot experiments were used to detected the expression of Ascl2,luciferase experiments were used to the activity change of transfection of luciferase reporter containing Ascl2 promoter into CD133+CD44+ and CD133-CD44-cells.Results1.Mucin-type O-glycans in regulating bacteria adherence to and invasion into intestinal epithelial cells1.1 benzyl-α-GalNAc treated HT-29 or HT-29-Gal cells altered their O-glycosylation status.1.2 Lectin histochemical staining confrmed stronger staining with PNA,which labeled Galβ1,3 GalNAc(Core 1 structure)in HT-29-Gal-OBN and C2 Gn T2-sh2/HT-29 cells,compared with control cells.1.3 A comparison of the adherence of EPEC and EHEC O157:H7 to HT-29-Gal and HT-29 cells indicated that the differentiation of HT-29 cells led to a reduction in the adherence of EPEC and EHEC O157:H7.EPEC and EHEC O157:H7 adhesion decreas ed after the abrogation of O-glycan biosynthesis mediated by benzyl-a-GalNAc treatment.Core 2 O-glycan-deficient HT-29 cells induced by C2GnT2 knockdown had a significant reduction in EPEC and EHEC O157:H7 adhesion in C2GnT2-sh2/HT-29 cells compared with HT-29 and shRNA-Ctr/HT-29 cells.1.4 EPEC or EHEC O157:H7 invasion into HT-29 and its derived cells was based on the intracellular presence of GFP-labeled bacteria.The differentiation of HT-29 cells led to a reduction in EPEC internalization compared with HT-29 cells(P<0.01).EPEC or EHEC O157:H7 invasion into O-glycan-deficient cells(HT-29-OBN and HT-29-Gal-OBN)increased compared with control cells(HT-29 and HT-29-Gal).(P<0.05 and P<0.01).Core 2 O-glycan-defcient HT-29 cells underwent a signifcant increase in EPEC(P<0.01)or EHEC O157:H7(P<0.05)invasion compared with control cells.1.5 MUC2 expression in benzyl-α-GalNAc-treated HT-29 cells was significantly reduced but unchanged in C2 Gn T2-deficient HT-29 cells.EPEC or EHEC O157:H7 infection in C2GnT2-deficient HT-29 cells deteriorated the epithelial barrier function.The occludin expression in the shRNA-Ctr/HT-29 and C2GnT2-sh2/HT-29 cells after infection with EPEC or EHEC O157:H7 was pyknic and discontinuous at the cell surface compared with its continuous distribution of control cells.2.Mucin-type Core 3 O-glycans in regulating the epithelial-mesenchymal transition(EMT)-mesenchymal-epithelial transition(MET)plascity in colon cancer cells2.1 The reduced expression of Core 3 synthase correlates with metastasis to lymph nodes and distant organs,resulting in poor prognosis for colorectal cancer(CRC)patients.2.2 Core 3 synthase re-expression in colon cancer cells inhibits proliferation,migration,and invasion and also arrests the in vivo growth of the xenograft.2.3 Mucin-type Core 3 O-glycan was synthesized at the membrane-tethered MUC1 N-terminus due to Core 3 synthase expression in colon cancer cells.This further inhibited the translocation of MUC1-C to the nucleus,initiated p53 gene transcription that was dependent on the inhibition of MUC1-C nucleus translocation,activated p53-mediated mi R-200 c expression,and resulted in mesenchymal-epithelial transition(MET).2.4 Inhibition of MUC1 via si RNA in Core 3 synthase re-expressed colon cancer cells enhanced MET.Whereas,inhibition of p53 via siRNA and mi R-200 c via mi R-200 c inhibitor in Core 3 synthase re-expressed colon cancer cells promoted the epithelial-mesenchymal transition(EMT)in a reversible manner.2.5 Core 3 synthase mRNA levels and the p53 m RNA levels or mi R-200 c levels in the colon cancerous samples were positively correlated.3.Ascl2 autoregulation in CRC cells3.1 Exogenous Ascl2 in colon cancer cells activated the endogenous Ascl2 expression via a direct autoactivatory loop,including Ascl2 binding with its own promoter and transcriptional activation of Ascl2 gene.3.2 Ascl2 higher expression in human CRC cancerous tissues led to more enrichment in immunoprecipitated DNA in CRC cancerous sample than peri-cancerous mucosa.3.3 R-spondin1/Wnt activated Ascl2 expression dose-dependently in CD133+CD44+ CRC population,but not in CD133-CD44-CRC population.3.4 R-spondin1/Wnt treatment in CD133+CD44+ or CRC CD133-CD44-population exerted a different pattern of “stemness” maintenance which was defined by the alteration of “stemness” associated genes mRNA and protein expression(Bmi1,C-myc,Oct-4 and Nanog)and tumorsphere formation.Conclusions1.Core 2 mucin-type O-glycan is related to EPEC and EHEC O157:H7 adherence to human colon carcinoma HT-29 epithelial cells.2.Core 2 mucin-type O-glycan inhibits EPEC or EHEC O157:H7 invasion into HT-29 epithelial cells.3.Core 3 mucin-type O-glycan restoration in colorectal cancer cells promotes MUC1/p53/mi R-200c-dependent epithelial identity.4.R-spondin1/Wnt-enhanced autoregulation of Ascl2 controls self-renewal of CRC progenitor cells. |
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