The Role Of MUC2in The Inhibition Of Adhesion And Invasion Of E. Coli K1Strains To Intestinal Epithelial Cells By Probiotics

BackgroundMucins are large multifunctional glycoproteins whose primary functions are to protect and lubricate the surfaces of epithelial tissues lining ducts and lumens within the human body. Several lines of evidence also support the involvement of mucins in more complex biological processes, such as epithelial cell renewal and differentiation, cell signaling, and cell adhesion. Recent studies have uncovered the role of select mucins in the pathogenesis of cancer, underscoring the importance of a detailed knowledge about mucin biology. Under normal physiological conditions, the production of mucins is optimally maintained by a host of elaborate and coordinated regulatory mechanisms, thereby affording a well-defined pattern of tissue, time, and developmental state-specific distribution. However, mucin homeostasis may be disrupted by the action of environmental and/or intrinsic factors that affect cellular integrity. This results in an altered cell behavior that often culminates into a variety of pathological conditions. Deregulated mucin production has indeed been associated with numerous types of cancers and inflammatory disorders.MUC2is the major gel-forming mucin of the small and large intestines and is the main structural component of the mucus gel. Under normal physiological conditions, MUC2are100%expressed in intestinal mucosal epithelium and reduced expression in colorectal tumors. Ho et al have demonstrated that the expression of MUC2gene product is correlated well with methylation of the proximal region of the promoter, and in colorectal carcinoma cells it has been shown that suppression of the MUC2gene in vitro and in vivo is associated with methylation of the promoter region. Furthermore, MUC2expression in the gastric epithelium is thought to be regulated by promoter methylation, and two specific cytosine guanine dinucleotide(CpG) sites may play a particularly regulatory role. Regulation of MUC2expression has been extensively studied, but the exact regulatory mechanism remains not fully understood.In addition, probiotics play an important role in strengthening the function of intestinal barrier, it can also contribute to the prevention and adjuvant treatment of neonatal infectious and allergic diseases. Studies have demonstrated, probiotics could induce MUC2secretion and up-regulate MUC2gene expression in human colorectal cancer cells, antagonize the adhesion and invasion of pathogens. Mattar et al. have reported that, compared with the control group, MUC2protein expression of the neonatal hirschsprung group was significantly reduced, and the children suffering from colitis with acute stage could not be detected MUC2expression. Preventive treatment with probiotics, such as Lactobacillus GG strain could increase the expression of MUC2to reduce bacterial translocation.However, the interactions between probiotics, pathogens and mucin gene is still not completely understood. The mechanism of MUC2in pathogen invasion of intestinal barrier remains unclear. Therefore, the aim of this study was to investigate the relationship between MUC2expression and the inhibition of probiotics in the adherence and invasion of E. coli K1strains. We constructed MUC2-targeted shRNA plasmid expression vector, and MUC2shRNA was subsequently transftected in to Caco-2cells. We evaluated the effect of inhibition of probiotics in the adherence and invasion of E. coli K1(E44) strains. One the other hand, we detected MUC2gene methylation status in Caco2cells and explored the possibility of re-expression of hypermethylated and silenced MUC2gene by5-Aza-CdR. The ability of5-Aza-CdR to interfere with the adhesion and invasion of E. coli K1(E44) to Caco-2cells was examined by Adhesion and invasion assays.Methods1. Silencing vectors construction and determinationKnockdown of MUC2was achieved by expression of short hairpin RNA (shRNA) from the pGPU6/GFP/Neo vector containing human U6promoter. The sequence of the oligonucleotide targeted to MUC2and the scrambled shRNA were designed and synthesized and pGPU6/GFP/Neo plasmid was linearized with BamH I and Bbs I to permit the insertion of the annealed oligonucleotides. The recombinant constructs were verified by analyzing the fragments generated from digestion with BamH I and by DNA sequencing. The interference efficiency was analyzed by RT-qPCR.2. Generation of transient transfectantCaco-2cells were seeded in12-well plates to80%～90%confluence. The cells were transfected with mixtures of shRNA plasmids and LipofectamineTM2000reagent (Invitrogen, USA) according to the manufacturer’s instructions.48h after transfection, transfected cells were observed via fluorescence microscope to estimated the transfection rate. Cells were also transfected with siRNA oligos containing no homology with any human gene as a negative control. 3. RN A extraction and reverse transcription PCRTransient transfectant clones with low expression of MUC2were evaluated by RT-qPCR analysis. Total RNA in Caco-2cells was extracted with Trizol (Invitrogen, USA), and the expression of MUC2mRNA was detected by RT-qPCR. The RNA expression level was represented as the ratio of the amount of MUC2over β-actin. Using2’AACt to calculate the relative expression of MUC2to filter out the most effective interference sequence which suppressed MUC2gene expression. In this analysis, data from three separate experiments were averaged.4. Adhesion and invasion AssaysFor the co-treatment experiments (competition assay), the cells were subcultured into24-well plates and then preincubated at37℃with108CFU of probiotics and107CFU of E44in the complete experimental medium for2.5h to allow adhesion and invasion to occur.For adhesion assays, the cells were washed three times with medium to remove unattached bacteria. The bacteria bound to intestinal mucus were released and lysed with200μL of0.5%TritonX-100for8min followed immediately by the addition of250μL of sterile water. This concentration of TritonX-100did not affect bacterial viability for at least30minutes (data not shown). Samples were diluted and plated onto LB agar plates with rifampin to determine the number of colony forming units (CFUs) recovered from the lysed cells. Each experiment was carried out in triplicate, and results presented are representative of the repeated assays. Results were expressed either as relative adhesion (percent adhesion as compared to the adhesion of the parent E44strain).For invasion assays, the cells were washed three times with medium, the number of intracellular bacteria was determined after the extracellular bacteria were eliminated by incubation of the monolayers with the experimental medium containing gentamicin(100μg/mL) for1h at37℃. Each experiment was carried out in triplicate and results were expressed either as relative invasion(percent invasion as compared to the invasion of the parent E44strain).5. RT-qPCR analysis after DNA methylation inhibitor(5-Aza-CdR) and bacterial treatment/infection assaysCaco-2cells were treated with5×10-6mol/L5-Aza-CdR (freshly prepared) respectively for72h. The medium was changed with the same concentration every24h. The cells with no5-Aza-CdR treatment were the control group, which treated with an equal volume of medium instead of5-Aza-CdR culture medium. Washed the well with sterile PBS for2times and added0.5mL/well experimental media without antibiotics before bacterial treatment/infection. Cells were incubated with probiotics (108CFU) and/or E44(107CFU) in the complete experimental medium for2.5h to allow adhesion and invasion to occur.Total RNA was extracted as described above. RT-qPCR were used to detect re-expression of MUC2mRNA before and after treatment with5-Aza-CdR respectively.6. Bacterial adhesion and invasion assays in5-Aza-CdR-treated Caco-2cellsThe adhesion and invasion assays in5-Aza-CdR-treated Caco-2cells were performed as described above.Results1. Restriction enzyme digestion and DNA sequencing of constructed vector Target DNA was cloned into pGPU6/GFP/Neo plasmid by T4DNA ligase. The recombinant constructs were verified by analyzing the fragments generated from digestion with BamH I and Pst I, respectively. The results showed that all plasmids were positive recombinant vectors and DNA sequencing results provided further confirmation of the presence of the recombinant plasmids, indicating that all the shRNA expression plasmids carried the correct sequence (data not show).2. Estimation of transfectant efficiencyCaco-2cells were transfected with mixtures of shRNA plasmids using LipofectamineTM2000and serum-free medium(Opti-MEM) according to the manufacturer’s recommendations. All transfections were done in triplicate and cells were harvested48h post-transfection. Transfection efficiency was typically75%-85%for synthetic siRNA by fluorescence microscopy.3. The vector expressing MUC2shRNA caused effective and specific down regulation of MUC2expressionIn order to study the potential role of MUC2in Caco-2cells, we used shRNA MUC2to selectively reduce MUC2expression in Caco-2cells. MUC2shRNA and scrambled shRNA plasmids were used to transfect Caco-2cells. Knockdown efficiencies were subsequently tested by RT-qPCR. MUC2mRNA levels were reduced by62%,41%、-0.8%、54%in Caco-2shRNA transfected cells, as compared with the control cells. The best knockdown efficiency is MUC2-1shRNA sequence. The scrambled shRNA sequence had no effect on MUC2expression. These results demonstrated that the expression of MUC2was specifically silenced in Caco-2cells.4. Inhibition of probiotics in E44adhesion to and invasion of Caco-2cells after MUC2silencingThe ability of probiotics to interfere with the adhesion and invasion of E. coli K1 to Caco-2cells was examined by competitive exclusion assays. In this study, Caco-2cells were preincubated with probiotics(108CFU) and E44(107CFU). Probiotics was able to competitively inhibit E44invasion and adhesion in Caco-2cells(P<0.05). Contrarily, after MUC2silencing, the inhibition of probiotics was significantly lower than the untreated group(P<0.01). The relative adhesion rate of E44is63.64%, and the relative invasion rate of E44is70.33%. The invasion and adhesion rate of E44at the zero concentration of probiotics was assigned as100%and the effects of probiotics preincubation were compared to this control level.5. Re-expression of MUC2in5-Aza-CdR-treated Caco-2cellsWe used5-Aza-CdR to selectively up-regulate MUC2expression in Caco-2cells. RT-qPCR analysis was shown that, the promoter region of MUC2exhibited a demethylation state, and induced MUC2re-expression. In5-Aza-CdR and probiotics group, MUC2expression was significantly up-regulated, however, in E44group, MUC2expression was lower than the control group(P<0.05).6. Effects of5-Aza-CdR on E44adhesion to and invasion of Caco-2cellsAfter treatment with5-Aza-CdR for72h, the ability of5-Aza-CdR and probiotics to interfere with the adhesion and invasion of E. coli K1to Caco-2cells was examined by competitive exclusion assays.5-Aza-CdR was able to competitively inhibit E44adhesion and invasion in Caco-2cells. The relative adhesion rate of E44was10.33%and the relative invasion rate of E44was29.45%. The result of probiotics group was similar to5-Aza-CdR treatment(P<0.05). The adhesion and invasion rate of E44at the zero concentration of probiotics and5-Aza-CdR was assigned as100%and the effects of5-Aza-CdR and probiotics preincubation were compared to this control level. Conclusion1. The expression of MUC2gene in Caco-2cells could be suppressed efficiently by RNA interference with eukaryotic expression plasmid containing shRNA.2. After silencing MUC2, the inhibition of probiotics in the adherence and invasion of E44strains to intestinal epithelial cells was significantly reduced.3. MUC2gene can be reversed by demethylation agent5-Aza-CdR which can regulate the expression of MUC2gene.4.5-Aza-CdR can strongly suppress E44penetration across Caco-2cells. We presume that5-Aza-CdR might play a protective role as probiotics in excluding pathogens from the intestine and preventing infections.