MUC1 Protein Expression Research Articles

Attempt to Substitute the Oncotype DX Breast Recurrence Score® Test by Histopathological Factors and MUC1 Protein Expression.

Oncotype DX Breast Recurrence Score® test (ODx) is a gene profiling assay predicting the benefit of adjuvant chemotherapy for early-stage hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Meanwhile, to avoid unnecessary financial burden on the patient, many studies have attempted to establish alternatives to ODx using conventional clinicopathological factors, but these have not yet been successful. Thus, we retrospectively investigated clinicopathological factors to establish alternatives to ODx. Data from 114 Japanese women who underwent ODx were retrospectively examined to investigate the relationship between ODx recurrence score (RS) and clinicopathological features, including MUC1 staining patterns on immunohistochemical assessment. An RS of 0-25 was defined as low, and 26-100 as high. Ninety patients (79%) had low RS and 24 patients (21%) had high RS. Univariate analysis revealed that low tumor grade, high progesterone receptor (PgR) expression, and low Ki67 labeling index (LI) were significantly associated with low RS (p=0.025, p<0.001, and p<0.001, respectively). Tumors with an apical pattern of MUC1 staining also frequently had a low RS (p=0.024). In multivariate analysis, PgR expression and Ki67 LI were independent factors associated with RS (p<0.001, for both). When the ODx results were categorized with a combination of these two factors, only 2% of the PgR-high and Ki67-low group (one in 51 cases) had a high RS. PgR expression and Ki67 LI were independent factors correlated with RS. MUC1 staining pattern also has the potential to be a useful marker. We believe that it is crucial to continue attempts to identify patients who are unlikely to benefit from ODx.

Lycium barbarum polysaccharide remodels colon inflammatory microenvironment and improves gut health

AimDisturbed intestinal microbiota has been implicated in the inflammatory microenvironment of the colon, which usually results in ulcerative colitis (UC). Given the limitations of these drugs, it is important to explore alternative means of protecting the gut health from UC. This study aimed to investigate the potential of polysaccharides as beneficial nutrients in the regulation of the gut microbiota, which determines the inflammatory microenvironment of the colon. Materials and methodsMice were treated with dextran sulfate sodium (DSS) to evaluate the effects and mechanisms of Lycium barbarum polysaccharide (LBP) in remodeling the inflammatory microenvironment and improving gut health. Body weight and disease activity indices were monitored daily. Hematoxylin and eosin staining was used to analyze colon dynamics. The levels of inflammatory indicators and expression of MUC-2, claudin-1, ZO-1, and G-protein-coupled receptor 5 (TGR5) were determined using assay kits and immunohistochemistry, respectively. 16S rRNA high-throughput sequencing of the intestinal microbiota and liquid chromatography-tandem mass spectrometry for related bile acids were used. ResultsLBP significantly improved the colonic tissue structure by upregulating MUC-2, claudin-1, and ZO-1 protein expression. The bacterial genus Dubosiella was dominant in healthy mice, but significantly decreased in mice treated with DSS. LBP rehabilitated Dubosiella in the sick guts of DSS mice to a level close to that of healthy mice. The levels of other beneficial bacterial genera Akkermansia and Bifidobacterium were also increased, whereas those of the harmful bacterial genera Turicibacter, Clostridium_sensu_stricto_1, Escherichia-Shigella, and Faecalibaculum decreased. The activity of beneficial bacteria promoted the bile acids lithocholic and deoxycholic acids in mice with UC, which improved the gut barrier function through the upregulation of TGR5. ConclusionThe inflammatory microenvironment in the gut is determined by the balance of the gut microbiota. LBP showed great potential as a beneficial nutrient for rehabilitating Dubosiella which is dominant in the gut of healthy mice. Nutrient-related LBP may play an important role in gut health management.

Effect of Cordyceps militaris Powder Prophylactic Supplementation on Intestinal Mucosal Barrier Impairment and Microbiota-Metabolites Axis in DSS-Injured Mice.

Ulcerative colitis (UC) is a chronic and recurrent inflammatory disease with an unknown pathogenesis and increasing incidence. The objective of this study is to investigate the impact of prophylactic treatment with Cordyceps militaris on UC. The findings demonstrate that prophylactic supplementation of C. militaris powder effectively mitigates disease symptoms in DSS-injured mice, while also reducing the secretion of pro-inflammatory cytokines. Furthermore, C. militaris powder enhances the integrity of the intestinal mucosal barrier by up-regulating MUC2 protein expression and improving tight junction proteins (ZO-1, occludin, and claudin 1) in DSS-injured mice. Multiomics integration analyses revealed that C. militaris powder not only reshaped gut microbiota composition, with an increase in Lactobacillus, Odoribacter, and Mucispirillum, but also exerted regulatory effects on various metabolic pathways including amino acid, glyoxylates, dicarboxylates, glycerophospholipids, and arachidonic acid. Subsequent analysis further elucidated the intricate interplay of gut microbiota, the intestinal mucosal barrier, and metabolites, suggesting that the microbiota-metabolite axis may involve the effect of C. militaris on intestinal mucosal barrier repair in UC. Moreover, in vitro experiments demonstrated that peptides and polysaccharides, derived from C. militaris, exerted an ability to change the gut microbiota structure of UC patients' feces, particularly by promoting the growth of Lactobacillus. These findings suggest that regulatory properties of C. militaris on gut microbiota may underlie the potential mechanism responsible for the protective effect of C. militaris in UC. Consequently, our study will provide support for the utilization of C. militaris as a whole food-based ingredient against the occurrence and development of UC.

Lyophilization Based Isolation of Exosomes.

Exosomes are nanoscale extracellular vesicles which regulate intercellular communication. They have great potential for application in nanomedicine. However, techniques for their isolation are limited by requirements for advanced instruments and costly reagents. In this study, we developed a lyophilization-based method for isolating exosomes from cultured cells. The isolated exosomes were characterized for protein content using Bradford assay, and for size distribution and shape using scanning electron microscopy (SEM) and nanoparticles tracking analysis (NTA). In addition, CD63, CD9, CD81, HSP70 and TSG101 were evaluated as essential exosomal surface markers using Western blot. Drug loading and release studies were performed to confirm their drug delivery properties using an in vitro model. Exosomes were also loaded with commercial dyes (Cy5, Eosin) for the evaluation of their drug delivery properties. All these characterizations confirmed successful exosome isolation with measurements of less than 150 nm, having a typical shape, and by expressing the known exosome surface protein markers. Finally, tyrosine kinase inhibitors (dasatinib and ponatinib) were loaded on the exosomes to evaluate their anticancer effects on leukemia cells (K562 and engineered Ba/F3-BCR-ABL) using MTT and Annexin-PI assays. The expression of MUC1 protein on the exosomes isolated from MCF-7 cells also indicated that their potential diagnostic properties were intact. In conclusion, we developed a new method for exosome isolation from cultured cells. These exosomes met all the essential requirements in terms of characterization, drug loading and release ability, and inhibition of proliferation and apoptosis induction in Ph+ leukemia cells. Based on these results, we are confident in presenting the lyophilization-based exosome isolation method as an alternative to traditional techniques for exosome isolation from cultured cells.

Low Expression of Mucin 2, High Expression of Mucin 13, and High Expression of Nuclear Factor Kappa-Light-Enhancer of Activated B Cells were Significant Pathways in Colorectal Cancer Development.

To prove the role of MUC2, MUC13, and NFκB protein expression as significant carcinogenesis pathways in colorectal cancer development. This is a nested case-control study conducted at the Udayana University, Sanglah General hospital, from September 2020 to 2021. All eligible subjects who visited the Digestive Surgery outpatient clinic with a colorectal surgery plan 2021 were included. The subjects were classified as case group (cancerous colonic mucosa) and control group (normal colonic mucosa), proved by histopathology examination. The parameters in this study were the expression of MUC2, MUC13, and NFκB by immunohistochemistry analysis. The data in this study will be collected and tabulated in SPSS 25.0 (Chicago, Illinois, USA). A total of 36 subjects with colorectal cancer (case group) and 36 subjects with normal colonic mucosa (control group) were analyzed in this study. The cancerous colonic mucosa significantly had a lower MUC2, higher MUC13, and higher NFκB expression. After multivariate analysis for controlling the age variable, the result showed that only MUC2, MUC13, and NFκB expressions were still significant with p<0.05. The effect from MUC2, MUC13, and NFκB expression totally could assess up to 85.4% of the risk of developing colorectal cancer. There was a significantly lower MUC2, higher MUC13, and higher NFκB expression in the carcinogenesis of colorectal cancer, representing the influence of the inflammatory pathway and the abnormality of the protective barrier. Therefore, in the future, this result could remark a future early prediction or scoring system to assess colorectal cancer in clinical application.

Prevention of Inflammation-Driven Colon Carcinogenesis in Human MUC1 Transgenic Mice by Vaccination with MUC1 DNA and Dendritic Cells

The preventive efficacy of MUC1-specific DNA immunization on inflammation-driven colon carcinogenesis in human MUC1 transgenic (MUC1.Tg) mice was investigated. Mice were vaccinated with MUC1 DNA mixed with autologous bone-marrow-derived dendritic cells (BMDCs), and then colonic tumors were induced by azoxymethane (AOM) injection and oral administration of dextran sulfate sodium (DSS). Two types of tumors, squamous metaplasia and tubular adenoma, were observed. Both expressed high levels of MUC1 as indicated by the binding of anti-MUC1 antibodies with different specificities, whereas MUC1 expression was not detected in normal colonic mucosa. When mice were immunized with MUC1 DNA + BMDCs, tumor incidence, tumor number, and tumor size were significantly reduced. In contrast, vaccination with MUC1 DNA alone or BMDCs alone was ineffective in reducing tumor burden. Inflammation caused by DSS was not suppressed by the MUC1 DNA + BMDCs vaccination. Furthermore, MUC1 protein expression levels, as judged by anti-MUC1 antibody binding in tumors grown after vaccination, did not significantly differ from the control. In conclusion, an inflammation-driven carcinogenesis model was established in MUC1.Tg mice, closely resembling human colon carcinogenesis. In this model, vaccination with MUC1 DNA + BMDCs was effective in overriding MUC1 tolerance and reducing the tumor burden by a mechanism not affecting the level of colonic inflammation.

Protective effect of sodium butyrate on intestinal barrier damage and uric acid reduction in hyperuricemia mice

PurposeThe goal of this study was to examine the role of sodium butyrate in preserving the intestinal mucosal barrier and reducing hyperuricemia (HUA). MethodsFirst, we established a mouse model of HUA via intraperitoneal injection of potassium oxonate together with a yeast-rich diet to detect the levels of serum uric acid (UA) and fecal short-chain fatty acids (SCFAs). Then, in vitro, different concentrations of UA and sodium butyrate (NaB) were used to treat LS174T and Caco2 cells. The effects of UA and NaB on the gut barrier were determined based on the expression levels of MUC2, ZO-1, and Occludin.Finally, C57BL/6 mice were used to model HUA, and these mice were administered 200 mg·kg−1·d−1 NaB by gavage to counter the HUA. The effect of NaB on HUA in the intestinal tract was elucidated by determining serum UA levels, inflammatory parameters, epithelial barrier integrity, and via histological analysis. ResultsThe data showed that the content of fecal SCFAs in HUA mice decreased. Additionally, in LS174T and Caco2 cells, NaB reversed the decrease of ZO-1, Occludin, and MUC2 protein expression caused by high UA levels. Furthermore, NaB decreased serum UA of HUA mice, and reversed both the decreased expression of MUC2, ZO-1, Occludin, and ABCG2 proteins and the increased level of inflammatory factors in the intestinal tissues of these mice. ConclusionThe HUA mouse model showed intestinal barrier damage. NaB protected the intestinal barrier of HUA mice and reduced the serum UA level.

Effect of electroacupuncture on Notch/NF-κB signaling pathway in colonic mucosa of mice with ulcerative colitis

To observe the protective effect of electroacupuncture (EA) on the intestinal mucosal barrier and its relationship with the Notch/NF-κB signaling pathway in mice with ulcerative colitis (UC), so as to explore its mechanism of treating UC. Male C57BL/6J mice were randomized into control, model and EA groups, with 6 mice in each group. The UC model was established by giving the mice with 2% Dextran Sulfate Sodium (DSS) for 7 days. EA (2 Hz/15 Hz, 0.2 mA) was applied at bilateral "Zusanli" (ST36) for 30 min, once a day for 7 days. The disease activity indexes ［DAI=(body weight index score+stool score+bleeding score)/3; 0-4 points］ of mice were calculated. The morphological changes of colonic tissues of mice in each group were observed by HE staining, and serum contents of TNF-α and IL-6 were detected by ELISA. Claudin-1 protein expression in colon tissue was detected by immunofluorescence, while the protein expression levels of Muc-2, Notch-1, MMP-9 in colon tissue were detected by immunohistochemistry. The real-time PCR method was used to detect the expression levels of Notch-1, Hes-1, NF-κB, TLR-4 and AKT mRNA in colon tissues. After modeling, the DAI, serum TNF-α and IL-6 contents, Notch-1 and MMP-9 protein expression, the relative expression levels of Notch-1, Hes-1, NF-κB, TLR-4 and AKT mRNA in the colonic tissue were significantly increased (P<0.001, P<0.01) in the model group relevant to the control group. At the same time, Claudin-1 and Muc-2 protein expression were significantly reduced (P<0.01). After the EA intervention, the increased DAI score, TNF-α and IL-6 contents, Notch-1 and MMP-9 protein expression, the relative expressions of Notch-1, Hes-1, NF-κB, TLR-4 and AKT mRNA, and the decreased Claudin-1 and Muc-2 protein expression were all reversed compared with the model group (P<0.05, P<0.01, P<0.001). H.E. staining of the colonic tissue showed damage and infiltration of inflammatory cells in the model group, and those were significantly improved in the EA group. EA can promote the recovery of intestinal mucosal barrier function and reduce inflammatory reaction in UC mice, which may be associated with its effects in inhibiting the excessive activation of the Notch/NF-κB signaling pathway.

Effect of smoking on MUC1 expression in oral epithelial dysplasia, oral cancer, and irradiated oral epithelium

ObjectivesThe aim of this study was to assess the MUC1 expression in the oral epithelium of normal, oral epithelial dysplasia (OED), oral squamous cell carcinoma (OSCC), and irradiated oral epithelium (IROE) and its association with smoking habits in non-smokers and smokers. DesignOral mucosal biopsies from controls, OED, OSCC, and IROE groups were obtained and categorized based on the smoking history as non-smokers, smoker I (25 pack-years), and smoker II (>25 pack-years). Immunohistochemical staining of MUC1 using human milk fat globule 1 (HMFG 1) antibody was performed, and the MUC1 score was calculated. The relation between MUC1 expression and clinicopathological findings was examined. ResultsMUC1 staining of superficial oral epithelial cells with mild MUC1 score was detected in all control samples. The MUC1 staining extended from superficial to basal cell layer of oral epithelium with the increase in MUC1 score from moderate to strong in OED, OSCC, and IROE, and the difference was significant (p < 0.004, p < 0.002 and p < 0.004, respectively) compared to controls. A positive association between smoking and MUC1 score was observed within groups (p < 0.05). ConclusionThe depolarization of MUC1 protein expression is associated with smoking habits in OED and OSCC. In the IROE, the radiation causes subcellular and molecular changes, observed as altered MUC1 expression and accelerated by smoking, furthermore, complicating the oral mucosal adaptation and progress to radiation-induced lesions as a delayed effect.

Abstract 6367: Activation of vitamin D/VDR signaling reverses gemcitabine resistance of pancreatic cancer cells through inhibition of MUC1 expression

Abstract Objective: Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis, largely due to its resistance to currently available therapies. Identifying the determinants of and signaling pathways regulating this resistance is essential for the development of effective therapeutic modalities to improve the outcome of PDA. The purpose of this study is to define the role of vitamin D/VDR signaling and underlying mechanisms in PDA pathogenesis and therapeutic resistance. Materials &amp; Methods: We used an unbiased reverse phase protein array (RPPA) to identify Vitamin D3 downstream molecules; we also used a variety of cellular and molecular biological approaches to determine the impact of vitamin D/VDR signaling on the expression of MUC1 mucin and the cellular response to gemcitabine treatment in PDA cells. We used in vivo experimental systems to determine the effect of combination use of gemcitabine with Vitamin D analogue on pancreatic tumorigenicity. Additionally, we used ultra-high resolution mass spectrometry (HRMS) to analyze the relative abundance of intracellular dFdCTP in tumor tissue samples. Results: We identified that MUC1 protein expression was significantly reduced in AsPC-1 human pancreatic cancer cells after treatment with vitamin D3 or its analogue calcipotriol (Cal) by the RPPA analysis, which was further confirmed in MiaPaca-2 and Colo357 pancreatic cancer cells. VDR negatively regulated MUC1 expression in pancreatic cancer cells in both gain- and loss-of-function assays. We established gemcitabine (Gem) resistant AsPC-1-GemR and Colo357-GemR cells and found that AsPC-1-GemR and Colo357-GemR cells had much lower levels of VDR but higher levels of MUC1 protein expression compared to their parental cells, Vitamin D3 or its analogue significantly induced VDR and inhibited MUC1 expression in AsPC-1-GemR and Colo357-GemR cells, and sensitized the resistant cells to gemcitabine treatment. The results of siRNA knockdown experiments indicated that inhibition of MUC1 expression was essential for Cal’s sensitization of PDA cells to Gem treatment in vitro. In keeping with these findings, administration of the Vitamin D3 analogue, paricalcitol (PA) significantly enhanced the therapeutic efficacy of Gem compared to chemotherapy alone in xenograft and syngeneic mouse models of pancreatic cancer without any significant systemic toxicity. The improved combination treatment correlated with increased intratumoral concentration of dFdCTP, the biological active metabolite of gemcitabine. Conclusions: This study reveals a previously unidentified vitamin D/VDR-MUC1 signaling pathway involved in the regulation of gemcitabine resistance of PDA. Our results suggest that combinational therapies which include targeted activation of vitamin D/VDR signaling may improve the outcomes of patients with PDA. Citation Format: Daoyan Wei, Margarete Hafley, Liang Wang, Yi Liu, Peiying Yang, Xiangsheng Zuo, Robert S. Bresalier. Activation of vitamin D/VDR signaling reverses gemcitabine resistance of pancreatic cancer cells through inhibition of MUC1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6367.

Colonic MUC2 Mucin Is Stabilized By FCGBP To Resist Salmonella Enterica Invasion In Goblet Cells

MUC2 mucin produced by goblet cells form the colonic mucus bilayer as the first line of innate defense against pathogen invasion by forming a physical barrier between the lumen and the underlying single layer of mucosal epithelial cells. Goblet cells also produce a variety of proteins that are associated with the mucus layer. Of these proteins, FCGBP is of significant interest due to its structural similarities to MUC2 mucin with unknown functions. In this study, we elucidated the putative functions of FCGBP embedded in MUC2 mucin in response to Salmonella enterica adherence and invasion in human colonic goblet cells. We hypothesis that FCGBP is coordinately produced with MUC2 mucin to play an integral role in forming the protective mucus layer in innate host defense. The aims of the study are: 1) to determine if FCGBP alters the structural integrity of the mucus layer and 2) to quantify structural alterations of the mucus layer in S. enterica adherence and invasion of goblet cells.To investigate whether FCGBP impaired mucus barrier functions, two cell types were investigated: Wildtype LS174T (WT) MUC2 mucus‐producing goblet cells and LS174T cells with a missense mutation in FCGBP (FCGBP MS). To determine if FCGBP MS led to loss in barrier function in the mucus layer, S. enterica adherence and invasion, and 0.2, 1, and 2 µM fluorescent bead penetrability inoculated on WT and FCGBP MS monolayers were quantified. MUC2 and FCGBP mRNA and protein expression induced by S. enterica in WT and FCGBP MS goblet cells were analysed by RT‐PCR and Western blotting. Cell killing by S. enterica was determined by LDH assay. S. enterica induced pro‐inflammatory cytokines responses were analyzed by RT‐PCR and human focus 15‐plex cytokine and chemokine array.FCGBP MS cells exhibited significant loss in MUC2 mucus barrier function as quantified by fluorescent beads penetrability further towards the epithelial cell surface temporally as compared to WT cells independent of bead size. Adherence of live S. enterica was significantly increased in FCGBP MS but not WT cells and induced robust MUC2 mRNA expression and mucus secretion in a time‐dependent manner. Similarly, S. enterica elicited high expression of pro‐inflammatory cytokine mRNA and protein release in FCGBP MS as compared to WT cells. FCGBP MS were readily invaded by S. enterica that resulted in increased cell death as compared to WT cells.More S. enterica adhered, invaded, and killed FCGBP MS as compared to WT cells. Fluorescent beads penetrated the mucus layer closer to the cell surface in FCGBP MS cells. In WT goblet cells, FCGBP and MUC2 were coordinately upregulated in response to S. enterica. The concurrent increase in pro‐inflammatory cytokine expression in FCGBP MS cells in response to S. enterica suggests that bacteria directly interacted with the cell surface suggesting an impaired protective mucus layer. The overall trend for increased bacterial invasion, pro‐inflammatory response, and cell death in FCGBP MS demonstrates that FCGBP was critical in providing structural integrity of the mucus layer.

Effects of Maren Pills on the Intestinal Microflora and Short-Chain Fatty Acid Profile in Drug-Induced Slow Transit Constipation Model Rats.

Background: Slow transit constipation (STC) is becoming a common and frequently occurring disease in today’s society, and it is necessary to explore the safe and effective treatment of STC. Method: Our study aimed to investigate whether the laxative effect of Maren pills (MRW) is associated with the regulation of intestinal microflora and intestinal metabolism in the colon. Loperamide hydrochloride-induced STC rats received MRW intragastrically for two consecutive weeks to evaluate the laxative effect of MRW involving the regulation of intestinal microflora, intestinal metabolism, and 5-HT signaling pathway. Intestinal microflora was detected by 16s rDNA sequencing, intestinal metabolism of short-chain fatty acids (SCFAs) was detected by HPLC, and the 5-HT signaling pathway was detected by WB, ELISA, immunofluorescence, and immunohistochemical analysis. Results: Our results revealed that the treatments with MRW increased not only the body weight, 24-h fecal number, 24-h wet fecal weight, 24-h dry fecal weight, fecal water content, and the intestinal propulsion rate but also the colonic goblet cell number, colonic Muc-2 protein expression, and colonic mucus layer thickness in the STC model rats. Moreover, MRW activated the 5-HT pathway by increasing the levels of 5-HT, 5-HIAA, 5-HT4R, CFTR, cAMP, and PKA in the colon tissue of STC rats. The 16S rDNA sequencing results showed that MRW improved the colonic microflora structure in colonic contents of STC rats, mainly by increasing Lactobacillus and decreasing Prevotella. Finally, we found that MRW regulated the SCFA metabolism in the colonic contents of the STC rats, mainly by increasing the contents of acetic acid, propionic acid, and butyric acid; the relative abundance of Lactobacillus was positively correlated with either contents of acetic acid, propionic acid, and butyric acid, and the relative abundance of Clostridium was negatively correlated. Conclusion: Our study further showed that MRW could improve constipation in STC rats, and the mechanism may be by regulating the intestinal microflora structure and improving the metabolism of SCFAs.

A198 COLONIC MUC2 MUCIN IS STABILIZED BY FCGBP TO RESIST SALMONELLA TYPHIMURIUM INVASION IN GOBLET CELLS

BackgroundThe colonic mucus bilayer is the first line of innate defense against pathogen invasion in the gut by forming a physical barrier between the lumen and the underlying single layer of mucosal epithelial cells. While MUC2 mucin is the primary component of the mucus layer, it also contains several mucus associated proteins of which FCGBP is of significant interest due to its structural similarities to MUC2 with unknown functions. Here we elucidated the functions of FCGBP in MUC2 mucin in response to S. typhimurium ( St) adherence and invasion in human colonic goblet cells.AimsHypothesis: FCGBP is coordinately produced with MUC2 mucin and plays an important role in innate host defense. The specific aims are:1. To determine if FCGBP alters the structural integrity of the mucus layer2. To determine the role of FCGBP in St adherence and invasion of goblet cellsMethodsTo investigate whether FCGBP impaired mucus barrier functions, wildtype LS174T (WT) MUC2 mucus-producing goblet cells and LS174T cells with a missense mutation in FCGBP (FCGBP MS) were used. To determine if FCGBP MS led to loss in barrier function, St adherence and invasion, and 0.2, 1, and 2 μM fluorescent bead penetration through the mucus monolayers were quantified. MUC2 and FCGBP mRNA and protein expression induced by St were analysed by RT-PCR and Western blotting. St-induced pro-inflammatory cytokines responses were analyzed by RT-PCR and human focus 15-plex cytokine and chemokine array. Cell killing was enumerated by LDH assay.ResultsFCGBP MS cells exhibited significant loss in MUC2 mucus barrier function as quantified by fluorescent beads penetration closer towards the epithelial cell surface temporally as compared to WT cells independent of bead size. Adherence of St was significantly increased in FCGBP MS cells and induced robust MUC2 mRNA and protein expressions in a time-dependent manner. Similarly, St elicited robust expression of pro-inflammatory cytokine mRNA and protein release in FCGBP MS as compared to WT cells. FCGBP MS were readily invaded by St that resulted in increased cell death as compared to WT cells.ConclusionsLoss of function by FCGBP MS, but not in WT cells, showed increased penetration of fluorescent beads through the mucus layer that resulted in increased St adherence, invasion, and cell death. In WT cells, FCGBP and MUC2 were coordinately upregulated in response to St. The concurrent increase in pro-inflammatory cytokine expression in FCGBP MS cells in response to St suggests that bacteria directly interacted with the cell surface indicative of an impaired mucus layer. The overall trend for increased bacterial invasion, pro-inflammatory response and cell death in FCGBP MS demonstrates that FCGBP was critical in providing structural integrity and protective functions of the mucus layer.Funding AgenciesCIHR

Solamargine inhibits the growth of hepatocellular carcinoma and enhances the anticancer effect of sorafenib by regulating HOTTIP-TUG1/miR-4726-5p/MUC1 pathway.

Hepatocellular carcinoma (HCC) is one of the most common primary malignancies. Drug resistance has significantly prevented the clinical application of sorafenib (SF), a first‐line targeted medicine for the treatment of HCC. Solamargine (SM), a natural alkaloid, has shown potential antitumor activity, but studies about antitumor effect of SM are obviously insufficient in HCC. In the present study, we found that SM significantly inhibited the growth of HCC and enhanced the anticancer effect of SF. In brief, SM significantly inhibited the growth of HepG2 and Huh‐7 cells. The combination of SM and SF showed a synergistic antitumor effect. Mechanistically, SM downregulated the expression of long noncoding RNA HOTTIP and TUG1, followed by increasing the expression of miR‐4726‐5p. Moreover, miR‐4726‐5p directly bound to the 3′‐UTR region of MUC1 and decreased the expression of MUC1 protein. Overexpression of MUC1 partially reversed the inhibitory effect of SM on HepG2 and Huh‐7 cells viability, which suggested that MUC1 may be the key target in SM‐induced growth inhibition of HCC. More importantly, the combination of SM and SF synergistically restrained the expression of MUC1 protein. Taken together, our study revealed that SM inhibited the growth of HCC and enhanced the anticancer effect of SF through HOTTIP‐TUG1/miR‐4726‐5p/MUC1 signaling pathway. These findings will provide potential therapeutic targets and strategies for the treatment of HCC.

The Bile Acids Specifically Modulate Colonic MUC2 and Tight Junction Protein Expression in the Human Colon Cancer Cell Line

Abstract Objectives Alterations to mucin secretion and epithelial tight junctions can compromise the ability of the epithelium to act as a barrier for the host to prevent pathogenic attack. Bile acids are synthesized in hepatocytes and released into the intestine, further modified by gut bacteria. Although many studies have investigated the changes of intestinal bile acids in the pathogenesis of various immune disorders, there are few reports about its function in preventing or interventing the dysfunction of the intestinal barrier. In this study, we sought to investigate the effects of the colonic bile acids on MUC2 and tight junction protein expression, which are crucial to colonic barrier. Methods Regulation of MUC2 and tight junction protein expression was assayed in the human colon cancer LS174T and T84 cells. The cells were treated with deoxycholic acid (DCA), lithocholic acid (LCA), 3-oxo-DCA, 3-oxo-LCA, isoDCA and isoLCA (100 μM or 200 μM), respectively. Proliferation of the cells was investigated with the MTT assay. mRNA expression of MUC2, ZO-1, occludin, claudin1 were measured by RT-PCR. Nuclear bile acid receptor FXR and TGR5, toll-like receptors and TLR adaptor MyD88, and genes (CDX2, AGR2, MyD88) related to mucin synthesis and secretion were also measured. Results In comparison with the untreated control, DCA, 3-oxo-DCA, isoDCA and isoLCA (100 μM) significantly upregulated the ZO-1, occludin and bile acid receptor FXR gene expression in the T84 cell. LCA, 3-oxo-LCA and isoLCA upregulated MUC2 expression at 200 μM, but showed no significant effect at 100 μM. DCA only significantly upregulated MUC2 expression at 200 μM, but isoDCA upregulated MUC2 expression independent of concentration in the LS174T cell. The expression of CDX2, AGR2, MyD88 was consistent with MUC2. Conclusions Bile acids at various concentrations specifically modulate MUC2 and tight junction protein expression, and thereby alter the colonic barrier function. This regulatory effect of bile acids could be mediated by activating bile acid receptors FXR. Funding Sources This work was financially supported by Ministry of Science and Technology of China (10000 Talent Project).

Detection of MUC1 protein on tumor cells and their derived exosomes for breast cancer surveillance with an electrochemiluminescence aptasensor

As a kind of malignant tumor, breast cancer needs more simple and sensitive diagnosis methods. Exosomes, a novel cancer marker, have the potential to be used in cancer biopsy alternative surveillance. Thus, the determination of the correlation between cancer cells and their exosome composition is of significance. Herein, an electrochemiluminescence (ECL) aptasensor has been designed to detect the MUC1 protein expression on breast cancer cells and their derived exosomes. CD63 aptamer was immobilized on the electrode surface to capture the cells and exosomes, and Ru(bpy) 3 2+ @SiO 2 nanoparticles (Ru@SiO 2 NPs) modified with MUC1 aptamer could recognize specifically MUC1 protein on cells and exosomes. The amplified ECL signal from Ru@SiO 2 NPs exhibited linear responses in the range of 7.53 - 753 μg/mL with a limit of detection (LOD) of 0.83 μg/mL for MCF-7 cells, as well as linear range of 3.22 × 10 −4 - 156 μg/mL with a LOD of 2.73 × 10 −4 μg/mL for their exosomes. The expression of MUC1 protein on the surface of MDA-MB-231 and SK-BR-3 cells and their exosomes, were further analyzed, indicating the consistency of MUC1 expression of breast cancer cells and their exosomes. Furthermore, this aptasensor was applied to the determination of MUC1 protein on exosomes in serum samples of patient and normal control, demonstrating its potential for clinical cancer surveillance. • An electrochemiluminescence aptasensor was developed to detect MUC1 protein. • Ru(bpy) 3 2+ @SiO 2 nanoparticles was used to generate highly sensitive signals. • MUC1 protein expression level was represented by the number of cells and exosomes. • MUC1 protein expression on different cancer cells and their exosomes was analyzed. • The exosomal protein level may reflect that of their parent cells.

Tetrandrine Induces HL-60 Cell Differentiation

To investigate the differentiation of acute promyelocytic leukemia (APL) cells induced by Tetrandrine targeting MUC1. HL-60 cells at logarithmic growth phase were choosen as object of reaserch and were divided into four groups: control, ATRA, Tetrandrine and ATRA+Tetrandrine group. Cell morphologic changes in each group were observed by microscopy. The change of cell differentiation ability was analyzed by nitro-blue tetrazolium (NBT) reduction test. The expression of cell surface differentiation antigen CD11b was measured by flow cytometry. The expression of MUC1 protein was detected by Western blot. The differentiation of HL-60 cell could be induced by Tetrandrine. The NBT reduction-positive rate of ATRA+Tetrandrine group was significantly higher than that in ATRA group and Tetrandrine group (P<0.05). The percentage of CD11b positive cells in ATRA+Tetrandrine group(43.62％±1.38％) was significantly higher than that in Tetrandrine group(15.25％±2.11％), ATRA group (28.84％±7.53％) and control group(8.16％±1.01％) (P<0.05). The content of MUC1 protein in Tetrandrine group was significantly lower than that in control group and ATRA group (P<0.05). Tetrandrine and ATRA can synergize to promote the differentiation and maturation of HL-60 cells, and the mechanism may be related with MUC1 expression.

Effect of seasonal variance on intestinal epithelial barriers and the associated innate immune response of the small intestine of the Chinese soft-shelled turtles

It is conceivable that pathological conditions can cause intestinal barrier disruption and innate immune dysfunction. However, very limited information has been reported on the effect of seasonal variance on intestinal barriers and innate immunity. The present study was designed to investigate the seasonal variance in intestinal epithelial barriers and the associated innate immune response of turtle intestines during hibernation and nonhibernation periods. Goblet cells (GCs) demonstrated dynamic actions of the mucosal barrier with strong Muc2 protein expression during hibernation. However, weak Muc2 expression during nonhibernation was confirmed by immunohistochemistry, immunofluorescence and immunoblotting. Furthermore, light and transmission electron microscopy revealed that the hypertrophy of GCs resulted in the hypersecretion of mucus granules (MGs) and created a well-developed mucosal layer during hibernation. The absorptive cells (ACs), forming a physical barrier of tight junctions, and desmosomes were firmly anchored during hibernation. Conversely, during nonhibernation, the integrity of tight junctions, adherence junctions and desmosomes was noticeable expanded, causing increased paracellular permeability. As further confirmation, there was strong zonula occluden-1 (ZO-1) and connexins 43 (Cx43) protein expression during hibernation and weak ZO-1 and Cx43 expression during nonhibernation. Moreover, the expression level of the innate immune response proteins Toll-like receptors 2 and 4 (TLR2 and 4) were enhanced during hibernation and were reduced during nonhibernation. These results provide rich information about the seasonal fluctuations that interrupt intestinal epithelial barriers and innate immune response, which might be essential for protection and intestinal homeostasis.

Up-regulation of microRNA-136 induces apoptosis and radiosensitivity of esophageal squamous cell carcinoma cells by inhibiting the expression of MUC1

ObjectiveThis present study is performed to figure out the role of microRNA-136 (miR-136) in radiosensitivity of esophageal squamous cell carcinoma (ESCC) cells through the regulation of MUC1. MethodsSeventy-four ESCC patients were divided into radiotherapy sensitive group and radiotherapy resistance group. Colony formation assay and flow cytometry were used to test the radiosensitivity of radiotherapy resistant strain and parent strain. The expression of miR-136 between radiotherapy resistant strain and parent strain was detected by RT-qPCR, and the expression of miR-136 in Eca109 and TE-1 cells as well as Eca109-R and TE-1-R cells was detected after different doses of X-ray irradiation. Eca109 and TE-1 cells as well as Eca109-R and TE-1-R cells with overexpression of miR-136 or co-overexpression of miR-136 and MUC1 were constructed. Cell proliferation, colony formation and apoptosis was detected by CCK-8 assay, colony formation assay, and flow cytometry, respectively. ResultsThe expression of miR-136 in ESCC tissues was lower and MUC1 mRNA and protein expression was higher than that in adjacent normal tissues. The expression of miR-136 was negatively correlated with the expression of MUC1 mRNA in ESCC. Low expression of miR-136 and high expression of MUC1 were associated with tumor size, lymph node metastasis and distant metastasis. The expression of miR-136 increased while the expression of MUC1 decreased in the radiotherapy sensitive group of ESCC patients relative to the radiotherapy resistant group. The colony formation ability of radiation resistant cell line was stronger than that of parent cell line, and the apoptosis rate showed an opposite trend. Up-regulation of miR-136 reduced the survival rate, suppressed colony formation ability and induced apoptosis of ESCC cells under irradiation, which was reversed by upregulated MUC1. ConclusionThis study demonstrates that up-regulation of miR-136 induces apoptosis and radiosensitivity of ESCC cells by inhibiting the expression of MUC1.

Expression of inositol-requiring enzyme 1β is downregulated in azoxymethane/dextran sulfate sodium-induced mouse colonic tumors.

Inflammatory bowel disease (IBD) is a risk factor in colon cancer. Endoplasmic reticulum (ER) stress is associated with IBD and cancer. In the current study an azoxymethane (AOM) and dextran sulfate sodium (DSS)-induced mouse colonic tumor model was established to analyze the expression of ER stress chaperone molecules. Female C57BL/6 mice were intraperitoneally injected with 12 mg/kg AOM. On the 7th day following AOM injection, mice were treated with 1% DSS supplemented to the drinking water for 7 days, then followed by 14 days of normal drinking water. The cycle of 7 days DSS plus 14 days normal water was repeated twice and colonic tumors were evaluated for their number and size. Mice in the control group were injected with saline and received normal drinking water for the course of the experiment. mRNA levels of cytokines, inositol-requiring enzyme (IRE)1α and 1β, their downstream targets X-box binding protein (XBP)1u, XBP1s and mucin (MUC) 2 and interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-α were detected by reverse transcription-quantitative polymerase chain reaction. IRE1α, IRE1β and MUC2 protein expression was evaluated by immunohistochemistry, and IRE1α and IRE1β levels were further assessed by western blot analysis. It was observed that tumors developed in the distal colon of mice treated with AOM/DSS. IL-6, IL-8 and TNF-α mRNA levels were significantly increased in mice of the tumor group compared with mice of the control group. There were no significant differences in IRE1α mRNA and protein expression between the two groups and XBP1s mRNA levels were increased in the tumor compared with the control group. IRE1β and MUC2 mRNA levels were significantly decreased in the tumor compared with the control group (decreased by 42 and 30%, respectively). IRE1β and MUC2 proteins were predominately expressed in colonic epithelial cells and expression was decreased in the tumor compared with the control group. In conclusion, the downregulation of IRE1β and MUC2 may reduce the ability of colon tissues to resist inflammation, thus promoting the occurrence and development of colonic tumors.