| Background and ObjectiveIntestinal mucus barrier is one of the natural barrier against external damage, the maincomposition of the mucus is mucin, and the molecular mass of mucin is mainly composedof O-glycan chains, therefore, the function of mucin is closely related with the O-glycanchains. Recent studies have found that the alteration of mucin O-glycosylation changes isaccompanied with the carcinogenesis and regulation of proliferation, adhesion and invasionof tumor cells. Some O-glycan chains as bacterial surface adhesin linked carbohydrateligands, however, the role of O-glycan chains involved in the regulation of cell biologicalbehavior and mechanisms of interaction between bacteria and intestinal epithelial cellsremain largely unclear.Mucin-type O-linked glycosylation is initiated by the family of ppGalNAcTs thatutilize UDP-GalNAc as the nucleotide donor substrate to modify protein substrates.GalNAc-Ser/Thr residues is further elaborated by downstream glycosyltransferases togenerate a series of Core O-linked glycans. Core2O-glycan chains is mainly expressed inthe stomach and colon, therefore, we inhibited the synthesis of O-glycan chains bybenzyl-α-GalNAc to explore whether O-glycan was involed in the internalization of E.coliinto intestinal epithelial cells, we inhibited Core2O-glycan chains synthetase (C2GnT-2)by RNA interfence to explore its effect on molecular mechanism of Escherichia coliinvasion into intestinal epithelial cells and biological behavior change.Methods1. HT-29cells were selected for this study and its differentiated cell type (HT-29-Gal)were established.2. HT-29and its differentiated cell types were treated with O-glycan chains synthesisinhibitor（benzyl-N-acetyl-α-D-galactosaminide，benzyl-α-GalNAc）and these types werenamed as HT-29-Obn and HT-29-Gal-Obn.3. Fluorescence histochemical detection of lectins, including SNA, MAA, PNA, DBA, UEA-I, GSAII and ConA, was performed in HT-29, HT-29-Gal, HT-29-Obn andHT-29-Gal-Obn cells.4.HT-29, HT-29-Gal, HT-29-Obn and HT-29-Gal-obn cells were co-cultured withEPEC and EHEC O157:H7for2h, then the gentamicin(100ug/ml) was added in the culturemedium for2h to kill cellular surface-adherent bacteria, the invaded intestinal epithelialcells were treated with0.25%trypsin contain0.025%Triton X100to release the bacteria,finally, the serial dilution cell lysates were added in the bacteria plate, the cloning countingwas finished at the next day to reveal the numbers of invaded bacteria into the different celltypes (HT-29, HT-29-Gal, HT-29-Obn and HT-29-Gal-Obn cells).5. Efficiency of C2GnT-2RNA interference in HT-29cells was confirmed by qRT-PCRand Western blotting.6. Infected with EPEC or EHEC O157: H7or not, TEER values in shRNA-C2GnT-2/HT-29and shRNA-Ctr/HT-29cells were measured postconfluetly and ZO-1andocculudin were stained with immunohistochemstry.7. IL-6or IL-8was measured in EPEC or EHEC O157: H7infected shRNA-C2GnT-2/HT-29or shRNA-Ctr/HT-29Cells using ELISA.8. Lectin histochemistry, including SNA, MAA and PNA, was performed in HT-29,shRNA-Ctr/HT-29and shRNA-C2GnT-2/HT-29cells.9. The proliferation assay was examined using MTT method, the in vitro invasioncapability was measured using the transwell chamber, the migration ability of HT-29,shRNA-Ctr/HT-29and shRNA-C2GnT-2/HT-29cells were recorded after woundedmechanically.10. ZO-1, Occludin, E-cadherin and Caspase-3proteins in C2GnT-2interfered HT-29cells were detected by SDS-PAGE and Western blotting.Results;1. Fluorescence histochemical staining of lectins in HT-29, HT-29-Gal, HT-29-Obn andHT-29-Gal-obn cells showed that there was no significant difference of seven differentlectins between HT-29-Obn cells and HT-29cells. MAA was declined, whease SNA, PNAand GSAII were increased in HT-29-Gal-obn cells when comparing with the HT-29-Galcells. GSAII expression in HT-29-Gal cells was less than that in HT-29cells. The resutsindicated that there was an alteration of glycosylation pattern in HT-29-Obn and HT-29- Gal-Obn cells.2. Bacterial invasion assay showed the numbers of EPEC or EHEC O157: H7invasioninto HT-29-Obn cells and HT-29-Gal-Obn cells were significantly increased compared withHT-29and HT-29-Gal cells (P<0.05).3. The qRT-PCR and Western blotting confirmed that the expression of C2GnT-2expression at both protein and transcription levels was significantly reduced（P<0.01）afterC2GnT-2RNA interference.4.Immunocytochemistcal staining of MAA, SNA and PNA in HT-29, shRNA-Ctr/HT-29and shRNA-C2GnT-2/HT-29cells showed that MAA in shRNA-C2GnT-2/HT-29cells was decreased, whereas SNA was increased compared with HT-29and shRNA-Ctr/HT-29cells.5.TEER values were decreased in both shRNA-C2GnT-2/HT-29cells and shRNA-Ctr/HT-29cells after infected with EPEC or EHEC O157:H7, but TEER decrease inbacteria infected shRNA-C2Gn-T-2/HT-29cells was more obvious than bacteria infectedshRNA-Ctr/HT-29cells (P<0.05).6. Infected with EPEC or EHEC O157: H7or not, immunofluorescence stainingshowed that there was no significant difference in ZO-1expression level and its distribution,Occludin appeared pyknic and discontinous on the cell surface, but no significant differencein Occludin protein levels was found between shRNA-C2GnT-2/HT-29cells and shRNA-Ctr/HT-29cells.7. IL-6and IL-8was negative in the culture spent media in both shRNA-C2Gn-T-2/HT-29cells and shRNA-Ctr/HT-29cells with EPEC or EHEC O157:H7infection ornot.8. The proliferation rate, the ability of migration and invasion were increased inshRNA-C2Gn-T-2/HT-29cells compared with shRNA-Ctr/HT-29cells.9. No significant difference of ZO-1, Occludin and E-cadherin protein levels wasfound between shRNA-C2GnT-2/HT-29Cells and shRNA-Ctr/HT-29cells (P>0.05). Conclusion1. Inhibition of O-glycan chains synthesis in intestinal epithelial cells leads to thealteration of glycosylation pattern.2. Inhibition of O-glycan chains synthesis in intestinal epithelial cells leads to theirsusceptibity to bacteria invasion.3. Core2O-glycan chains synthesis inhibition does not affect the intestinal epithelialcell barrier, but the TEER value was more obviously reduced in shRNA-G2GnT2/HT-29than shRNA-Ctr/HT-29cells due to EPEC or EHEC O157:H7infection.4. EPEC and EHEC O157: H7infection in both shRNA-G2GnT2/HT-29cells andshRNA-Ctr/HT-29cells leads to the alteration of Occludin, appear as pyknic anddiscontinous.5. C2GnT-2RNA interference in HT-29cells leads to the increase of proliferation rate,migration and invasion ability. |
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