Different Forms of TFF3 in the Human Endocervix, including a Complex with IgG Fc Binding Protein (FCGBP), and Further Aspects of the Cervico-Vaginal Innate Immune Barrier.

TFF3 is a typical secretory poplypeptide of mucous epithelia belonging to the trefoil factor family (TFF) of lectins. In the intestine, respiratory tract, and saliva, TFF3 mainly exists as a high-molecular-mass complex with IgG Fc binding protein (FCGBP), which is indicative of a role in mucosal innate immunity. For the first time, we identified different forms of TFF3 in the endocervix, i.e., monomeric and homodimeric TFF3, as well as a high-molecular-mass TFF3-FCGBP complex; the latter also exists in a hardly soluble form. Immunohistochemistry co-localized TFF3 and FCGBP. Expression analyses of endocervical and post-menopausal vaginal specimens revealed a lack of mucin and TFF3 transcripts in the vaginal specimens. In contrast, genes encoding other typical components of the innate immune defense were expressed in both the endocervix and vagina. Of note, FCGBP is possibly fucosylated. Endocervical specimens from transgender individuals after hormonal therapy showed diminished expression, particularly of FCGBP. Furthermore, mucus swabs from the endocervix and vagina were analyzed concerning TFF3, FCGBP, and lysozyme. It was the aim of this study to illuminate several aspects of the cervico-vaginal innate immune barrier, which is clinically relevant as bacterial and viral infections are also linked to infertility, pre-term birth and cervical cancer.

Tff3 Deficiency Differentially Affects the Morphology of Male and Female Intestines in a Long-Term High-Fat-Diet-Fed Mouse Model.

Trefoil factor family protein 3 (Tff3) protects the gastrointestinal mucosa and has a complex mode of action in different tissues. Here, we aimed to determine the effect of Tff3 deficiency on intestinal tissues in a long-term high-fat-diet (HFD)-fed model. A novel congenic strain without additional metabolically relevant mutations (Tff3-/-/C57Bl6NCrl strain, male and female) was used. Wild type (Wt) and Tff3-deficient mice of both sexes were fed a HFD for 36 weeks. Long-term feeding of a HFD induces different effects on the intestinal structure of Tff3-deficient male and female mice. For the first time, we found sex-specific differences in duodenal morphology. HFD feeding reduced microvilli height in Tff3-deficient females compared to that in Wt females, suggesting a possible effect on microvillar actin filament dynamics. These changes could not be attributed to genes involved in ER and oxidative stress, apoptosis, or inflammation. Tff3-deficient males exhibited a reduced cecal crypt depth compared to that of Wt males, but this was not the case in females. Microbiome-related short-chain fatty acid content was not affected by Tff3 deficiency in HFD-fed male or female mice. Sex-related differences due to Tff3 deficiency imply the need to consider both sexes in future studies on the role of Tff in intestinal function.

TFF3 mediates the NF-κB/COX2 pathway to regulate PMN-MDSCs activation and protect against necrotizing enterocolitis.

Intestinal trefoil factor 3 (TFF3) plays an important role in repairing the intestinal mucosa. However, the detailed mechanism regarding immune regulation by TFF3 is not well defined. Here, we reported that treatment of mouse BM cells and human peripheral blood mononuclear cells from healthy volunteers with TFF3 activated polymorphnuclear myeloid-derived suppressor cells (PMN-MDSCs) in vitro. We also found that prostaglandin E2 is a major TFF3-mediated MDSC target, and that NF-κB/COX2 signaling was involved in this process. Moreover, TFF3 treatment or transfer of TFF3-derived PMN-MDSCs (TFF3-MDSCs) to experimental necrotizing enterocolitis (NEC) mice caused PMN-MDSC accumulation in the lamina propria (LP), which was associated with decreased intestinal inflammation, permeability, bacterial loading, and prolonged survival. Interestingly, no NEC severity remission was observed in Rag1 KO mice that were given TFF3-MDSCs, but coinjection with CD4+ T cells significantly relieved NEC inflammation. Overall, TFF3 mediates the NF-κB/COX2 pathway to regulate PMN-MDSC activation and attenuates NEC in a T-cell-dependent manner, which suggests a novel mechanism in preventing NEC occurrence.

Augmented leptin-induced trefoil factor 3 expression and epidermal growth factor receptor transactivation differentially influences neoplasia progression in the stomach and colorectum of dietary fat-induced obese mice.

Obesity is a risk factor for gastrointestinal malignancies and tumors. However, which factors either protect or predispose the gastrointestinal organs to high-fat diet (HFD)-induced neoplasia remains unclear. Here, we demonstrate that HFD impacts the stomach to a greater extent as compared to the colorectum, resulting in leptin receptor (LepR) signaling-mediated neoplasia in the tissues. HFD activated leptin signaling, which in turn, accelerates the pathogenesis in the gastric mucosa more than that in the colorectum along with ectopic TFF3 expression. Moreover, in the stomach, higher levels of phosphorylated epidermal growth factor receptor (EGFR) in addition to the activation of STAT3 and Akt were observed as compared to the colorectum. The mice with LepR deletion in the gastrointestinal epithelium exhibited a suppressed induction of leptin, TFF3, and phosphorylated EGFR in the stomach, whereas the levels in the colorectum were insignificant. In co-transfected COS-7 cells with LepR and EGFR plasmid DNA, leptin transactivated EGFR to accelerate TFF3 induction along with activation of STAT3, ERK1/2, Akt, and PI3K p85/p55. Furthermore, TFF3 could bind to EGFR but did not transactivate LepR. Leptin-induced TFF3 induction was markedly suppressed by inhibitors of PI3K (LY294002) and EGFR (Erlotinib). Together, these results suggest a novel role of LepR-mediated signaling in transactivating EGFR that leads to TFF3 expression via the PI3K-Akt pathway. Therefore, this study sheds light on the identification of potentially new therapeutic targets for the treatment of pre-cancerous symptoms in stomach and colorectum.

Low expression of TFF3 in papillary thyroid carcinoma may correlate with poor prognosis but high immune cell infiltration.

Background: Papillary thyroid carcinoma (PTC) is one of the most common endocrine malignancies and has a favorable prognosis. However, optimal treatments and prognostic markers have not been clearly identified. Methods: Gene expression data from primary PTC were downloaded from the Gene Expression Omnibus database and subjected to two analyses of differentially expressed genes (DEGs), followed by intersecting individual and integrated DEGs analyses as well as gene set enrichment analysis. Analysis of data from Sequence Read Archive and The Cancer Genome Atlas, immunohistochemistry and qRT-PCR of TFF3 were performed to validate the results. Finally, the relationship between gene expression and disease-free survival as well as immune cell infiltration were investigated. Results: Six critical DEGs and several tumor-enriched signaling pathways were identified. Immunohistochemistry and qRT-PCR validated the low expression of TFF3 in PTC. TFF3 and FCGBP are coexpressed in PTC, and patients with lower gene expression had worse disease-free survival but higher immune cell infiltration. Conclusion: TFF3 was significantly underexpressed and may function with FCGBP synergistically in PTC.

Lay abstract Thyroid cancers are some of the most common endocrine malignancies. However, the optimal treatments and prognostic markers have not been clearly identified. We identified six critical differentially expressed genes and several tumor-enriched signaling pathways in papillary thyroid carcinoma, and found that TFF3 was the most underexpressed gene, as validated by experiment. In addition, TFF3 and FCGBP worked synergistically and may mark prognosis and tumor immune cell infiltration, which may benefit patients with papillary thyroid carcinoma by providing early indication and prompting further basic investigation.

Correlation between CA12 and TFF3 and their prediction value of neoadjuvant chemotherapy response in breast cancer.

WHAT IS KNOWN AND OBJECTIVE: Compared with other molecular subtypes, hormone receptor-positive breast cancer often shows worse neoadjuvant chemotherapy efficacy. This study aims to explore the relationship between the oestrogen receptor (ER)-related genes carbonic anhydrase 12 (CA12) and trefoil factor 3 (TFF3) and their predictive value of neoadjuvant chemotherapy for breast cancer. METHODS: We investigated the relationships between CA12, TFF3 and ER status and their predictive value of anthracycline-taxane neoadjuvant chemotherapy in 115 female breast cancer patients via real-time polymerase chain reaction (RT-PCR) and 4 GEO datasets: GSE41998, GSE25065, GSE20194 and GSE20271. Then, the effects of CA12 and TFF3 on the chemotherapy drugs doxorubicin and docetaxel were verified in vitro in the breast cancer cell lines MCF-7 and BT474. RESULTS AND DISCUSSION: The GEO datasets and RT-PCR results showed that the relative expression of both CA12 and TFF3 was higher in oestrogen receptor-positive samples compared with the other samples (p < 0.05). CA12 was significantly correlated with TFF3 (p < 0.05). In MCF-7 cells, inhibition of TFF3 induced downregulation of CA12 and ESR1 (p < 0.05) at both the mRNA and the protein levels, while inhibition of CA12 also downregulated TFF3 and ESR1 (p < 0.05). In BT474 cells, inhibition of TFF3 downregulated CA12 and ESR1 (p < 0.05) at both the mRNA and the protein levels, while inhibition of CA12 led to slight upregulation of TFF3 and ESR1 (p > 0.05). Moreover, GEO datasets and RT-PCR results showed that CA12 and TFF3 were more highly expressed in nonpathological complete response (non-pCR) samples than in pCR samples (p < 0.05). Cell viability assays of MCF-7 and BT474 cells showed that inhibiting CA12 and TFF3 could enhance sensitivity to doxorubicin and docetaxel (p < 0.05). WHAT IS NEW AND CONCLUSION: CA12 and TFF3 were correlated with each other, and their high expression might explain the worse efficacy of neoadjuvant chemotherapy in oestrogen receptor-positive breast cancer patients.

Arsenic exposure in drinking water reduces Lgr5 and secretory cell marker gene expression in mouse intestines.

Arsenic is a global health concern that causes toxicity through ingestion of contaminated water and food. In vitro studies suggest that arsenic reduces stem and progenitor cell differentiation. Thus, this study determined if arsenic disrupted intestinal stem cell (ISC) differentiation, thereby altering the number, location, and/or function of intestinal epithelial cells. Adult male C57BL/6 mice were exposed to 0 or 100 ppb sodium arsenite (AsIII) through drinking water for 5 weeks. Duodenal sections were collected to assess changes in morphology, proliferation, and cell types. qPCR analysis revealed a 40% reduction in Lgr5 transcripts, an ISC marker, in the arsenic-exposed mice, although there were no changes in the protein expression of Olfm4. Secretory cell-specific transcript markers of Paneth (Defa1), Goblet (Tff3), and secretory transit amplifying (Math1) cells were reduced by 51%, 44%, and 30% respectively, in the arsenic-exposed mice, indicating significant impacts on the Wnt-dependent differentiation pathway. Further, protein levels of phosphorylated ß-catenin were reduced in the arsenic-exposed mice, which increased the expression of Wnt-dependent transcripts CD44 and c-myc. PCA analysis, followed by MANOVA and regression analyses, revealed significant changes and correlations between Lgr5 and the transit amplifying (TA) cell markers Math1 and Hes1, which are in the secretory cell pathway. Similar comparisons between Math1 and Defa1 show that terminal differentiation into Paneth cells is also reduced in the arsenic-exposed mice. The data suggests that ISCs are not lost following arsenic exposure, but rather, specific Wnt-dependent progenitor cell formation and terminal differentiation in the small intestine is reduced.

The diagnostic and clinicopathological value of trefoil factor 3 in patients with gastric cancer: a systematic review and meta-analysis.

OBJECTIVE: To assess the diagnostic value of Trefoil factor 3 (TFF3) and the correlation between TFF3 expression and clinicopathological features in patients with gastric cancer (GC). METHODS: PubMed, The Cochrane, EMbase, and Web of Science were retrieved comprehensively to collect relevant literature. The search ended on 31 May 2020. All data were analyzed using PubMed, The Cochrane, EMbase, and Web of Science were retrieved to collect relevant articles. All data from the included studies were subjected to meta-analysis using Stata 12.0 software. RESULTS: Seventeen studies involved 4654 subjects were included. For the diagnostic value of TFF3 for GC, the sensitivity, specificity, and AUC were 0.71, 0.80, and 0.80, respectively. For the clinicopathological value of TFF3, tissue TFF3 expression showed a higher risk of lymph node metastasis (OR 2.20, 95% CI 1.75-2.78, p < 0.001) and muscularis propria invasion (≥T2) (1.51, 1.13-2.03, p = 0.006), as well as worse TNM stage (2.26, 1.63-3.12, p < 0.001) and histological type (1.72, 1.34-2.20, p < 0.001), while no apparent relationship was found for serous membrane invasion (T4), venous invasion, and peritoneal metastasis. CONCLUSION: TFF3 may be a promising biomarker for GC, and the TFF3 expression is likely to be involved in the invasion, metastasis, and differentiation of GC.

LINC00160 mediated paclitaxel-And doxorubicin-resistance in breast cancer cells by regulating TFF3 via transcription factor C/EBPß.

Chemoresistance represents a major challenge in breast cancer (BC) treatment. This study aimed to probe the roles of LINC00160 in paclitaxel- and doxorubicin-resistant BC cells. Three pairs of BC and adjacent normal tissue were used for lncRNA microarray analysis. Paclitaxel-resistant MCF-7 (MCF-7/Tax) and doxorubicin-resistant BT474 (BT474/Dox) cells were generated by exposure of parental drug-sensitive MCF-7 or BT474 cells to gradient concentrations of drugs. Correlation between LINC00160 expression and clinical response to paclitaxel in BC patients was examined. Short interfering RNAs specifically targeting LINC00160 or TFF3 were designed to construct LINC00160- and TFF3-depleted BC cells to discuss their effects on biological episodes of MCF-7/Tax and BT474/Dox cells. Interactions among LINC00160, transcription factor C/EBPß and TFF3 were identified. MCF-7/Tax and BT474/Dox cells stable silencing of LINC00160 were transplanted into nude mice. Consequently, up-regulated LINC00160 led to poor clinical response to paclitaxel in BC patients. LINC00160 knockdown reduced drug resistance in MCF-7/Tax and BT474/Dox cells and reduced cell migration and invasion. LINC00160 recruited C/EBPß into the promoter region of TFF3 and increased TFF3 expression. LINC00160-depleted MCF-7/Tax and BT474/Dox cells showed decreased tumour growth rates in nude mice. Overall, we identified a novel mechanism of LINC00160-mediated chemoresistance via the C/EBPß/TFF3 axis, highlighting the potential of LINC00160 for treating BC with chemoresistance.

Mouse trefoil factor 3 ameliorated high-fat-diet-induced hepatic steatosis via increasing peroxisome proliferator-activated receptor-α-mediated fatty acid oxidation.

Hepatic trefoil factor 3 (Tff3) was identified as a potential protein for the treatment of diabetes, yet the effect of Tff3 on nonalcoholic fatty liver disease (NAFLD) has never been explored. Here, we found that the expression of hepatic Tff3 was significantly decreased in NAFLD mice models, suggesting that Tff3 was a potential marker gene for NAFLD. Restoring the expression of Tff3 in the liver of NAFLD mice, including diabetic (db), obese (ob/ob), and diet-induced obese mice, with adenovirus-mediated Tff3 (Ad-Tff3) apparently attenuates the fatty liver phenotype. In contrast, adenovirus-mediated knockdown of Tff3 (Ad-shTff3) in C57BL/6J mice results in an obvious fatty liver phenotype. Furthermore, our molecular experiments indicated that hepatic Tff3 could alleviate hepatic steatosis via upregulating the expression of peroxisome proliferator-activated receptor-α (PPARα) directly, thereby enhancing the fatty acid oxidation process in the liver. Notably, we found that Tff3 attenuates the fatty liver phenotype independent of modulation of lipogenesis and improves the capacity of anti-inflammation. Overall, our results suggested that hepatic Tff3 could be effectively used as a potential therapy target for the treatment of NAFLD.

Slug and Snail have differential effects in directing colonic epithelial wound healing and partially mediate the restitutive effects of butyrate.

Restitution of wounds in colonic epithelium is essential in the maintenance of health. Microbial products, such as the short-chain fatty acid butyrate, can have positive effects on wound healing. We used an in vitro model of T84 colonic epithelial cells to determine if the Snail genes Slug (SNAI2) and Snail (SNAI1), implemented in keratinocyte monolayer healing, are involved in butyrate-enhanced colonic epithelial wound healing. Using shRNA-mediated Slug/Snail knockdown, we found that knockdown of Slug (Slug-KD), but not Snail (Snail-KD), impairs wound healing in scratch assays with and without butyrate. Slug and Snail had differential effects on T84 monolayer barrier integrity, measured by transepithelial resistance, as Snail-KD impaired the barrier (with or without butyrate), whereas Slug-KD enhanced the barrier, again with or without butyrate. Targeted transcriptional analysis demonstrated differential expression of several tight junction genes, as well as focal adhesion genes. This included altered regulation of Annexin A2 and ITGB1 in Slug-KD, which was reflected in confocal microscopy, showing increased accumulation of B1-integrin protein in Slug-KD cells, which was previously shown to impair wound healing. Transcriptional analysis also indicated altered expression of genes associated with epithelial terminal differentiation, such that Slug-KD cells skewed toward overexpression of secretory cell pathway-associated genes. This included trefoil factors TFF1 and TFF3, which were expressed at lower than control levels in Snail-KD cells. Since TFFs can enhance the barrier in epithelial cells, this points to a potential mechanism of differential modulation by Snail genes. Although Snail genes are crucial in epithelial wound restitution, butyrate responses are mediated by other pathways as well.NEW & NOTEWORTHY Although butyrate can promote colonic mucosal healing, not all of its downstream pathways are understood. We show that the Snail genes Snail and Slug are mediators of butyrate responses. Furthermore, these genes, and Slug in particular, are necessary for efficient restitution of wounds and barriers in T84 epithelial cells even in the absence of butyrate. These effects are achieved in part through effects on regulation of ß1 integrin and cellular differentiation state.

Trefoil factors peptide-3 is associated with residual invasive breast carcinoma following neoadjuvant chemotherapy.

BACKGROUND: Breast carcinoma is the commonest cancer among UAE population and the most common cancer among females. Examination of the 5' promoter regions of trefoil factor 3 (TFF3) gene has identified putative estrogen and progesterone receptor-DNA binding domains as direct response elements to estrogen and progesterone that are linked to breast functions or steroid regulation. The study was designed to determine the role of TFF3 in breast cancer chemoresistance with the aim of establishing TFF3 expression as a biomarker for drug resistance. METHODS: In total, 133 cases of breast carcinoma treated with neo-adjuvant therapy were collected. Tissue samples from pre-neoadjuvant therapy as well as tissues from post-neo-adjuvant therapy of those cases were collected and stained with immunohistochemistry for TFF3, Bcl2, BAX, cleaved caspase-3, AKT-1, NF kappa B and Ki-67. RESULTS: There was increased expression of TFF3 in residual invasive carcinoma cells. There was a significant correlation between the expression of TFF3 in breast carcinoma cells and response to neoadjuvant chemotherapy (p = 0.0165). There was significant co-expression of TFF3 with AKT1 (p = 0.0365), BCl2 (p = 0.0152), and NF Kappa-B (p = 0.0243) in breast carcinoma cases with residual carcinoma following neoadjuvant therapy which support the role of TFF3 in chemoresistance. CONCLUSION: The expression of TFF3 is significantly associated with residual breast carcinoma following neoadjuvant chemotherapy suggesting its expression is associated with increased resistance to chemotherapy. This is supported by its co-expression with antiapoptotic proteins; BCl2, AKT1 and NF Kappa-B in residual breast carcinoma cells and very low proliferating index and apoptotic bodies in residual tumors.

Cathelicidin-WA Improves Intestinal Epithelial Barrier Function and Enhances Host Defense against Enterohemorrhagic Escherichia coli O157:H7 Infection.

Impaired epithelial barrier function disrupts immune homeostasis and increases inflammation in intestines, leading to many intestinal diseases. Cathelicidin peptides suppress intestinal inflammation and improve intestinal epithelial barrier function independently of their antimicrobial activity. In this study, we investigated the effects of Cathelicidin-WA (CWA) on intestinal epithelial barrier function, as well as the underlying mechanism, by using enterohemorrhagic Escherichia coli (EHEC)-infected mice and intestinal epithelial cells. The results showed that CWA attenuated EHEC-induced clinical symptoms and intestinal colitis, as did enrofloxacin (Enro). CWA decreased IL-6 production in the serum, jejunum, and colon of EHEC-infected mice. Additionally, CWA alleviated the EHEC-induced disruption of mucin-2 and goblet cells in the intestine. Interestingly, CWA increased the mucus layer thickness, which was associated with increasing expression of trefoil factor 3, in the jejunum of EHEC-infected mice. CWA increased the expression of tight junction proteins in the jejunum of EHEC-infected mice. Using intestinal epithelial cells and a Rac1 inhibitor in vitro, we demonstrated that the CWA-mediated increases in the tight junction proteins might depend on the Rac1 pathway. Furthermore, CWA improved the microbiota and short-chain fatty acid concentrations in the cecum of EHEC-infected mice. Although Enro and CWA had similar effects on intestinal inflammation, CWA was superior to Enro with regard to improving intestinal epithelial barrier and microbiota in the intestine. In conclusion, CWA attenuated EHEC-induced inflammation, intestinal epithelial barrier damage, and microbiota disruption in the intestine of mice, suggesting that CWA may be an effective therapy for many intestinal diseases.

Deoxynivalenol inhibits the expression of trefoil factors (TFF) by intestinal human and porcine goblet cells.

Trefoil factors (TFFs) are bioactive peptides expressed by several epithelia, including the intestine, where they regulate key functions such as tissue regeneration, barrier function and inflammation. Although food-associated mycotoxins, including deoxynivalenol (DON), are known to impact many intestinal functions, modulation of TFFs during mycotoxicosis has never been investigated. Here, we analyzed the effect of DON on TFFs expression using both human goblet cells (HT29-16E cells) and porcine intestinal explants. Results showed that very low doses of DON (nanomolar range) inhibit the secretion of TFFs by human goblet cells (IC50 of 361, 387 and 243 nM for TFF1, 2 and 3, respectively) and prevent wound healing. RT-qPCR analysis demonstrated that the inhibitory effect of DON is related to a suppression of TFFs mRNA expression. Experiments conducted on porcine intestinal explants confirmed the results obtained on cells. Finally, the use of specific inhibitors of signal pathways demonstrated that DON-mediated suppression of TFFs expression mainly involved Protein Kinase R and the MAP kinases (MAPK) p38 and ERK1/2. Taken together, our results show for the first time that at very low doses, DON suppresses the expression and production of intestinal TFFs and alters wound healing. Given the critical role of TFFs in tissue repair, our results suggest that DON-mediated suppression of TFFs contributes to the alterations of intestinal integrity the caused by this toxin.

Human Synovia Contains Trefoil Factor Family (TFF) Peptides 1-3 Although Synovial Membrane Only Produces TFF3: Implications in Osteoarthritis and Rheumatoid Arthritis.

OBJECTIVE: Trefoil factor family peptide 3 (TFF3) has been shown to support catabolic functions in cases of osteoarthritis (OA). As in joint physiology and diseases such as OA, the synovial membrane (SM) of the joint capsule also plays a central role. We analyze the ability of SM to produce TFF compare healthy SM and its secretion product synovial fluid (SF) with SM and SF from patients suffering from OA or rheumatoid arthritis (RA). METHODS: Real-time PCR and ELISA were used to measure the expression of TFFs in healthy SM and SM from patients suffering from OA or RA. For tissue localization, we investigated TFF1-3 in differently aged human SM of healthy donors by means of immunohistochemistry, real-time PCR and Western blot. RESULTS: Only TFF3 but not TFF1 and -2 was expressed in SM from healthy donors as well as cases of OA or RA on protein and mRNA level. In contrast, all three TFFs were detected in all samples of SF on the protein level. No significant changes were observed for TFF1 at all. TFF2 was significantly upregulated in RA samples in comparison to OA samples. TFF3 protein was significantly downregulated in OA samples in comparison to healthy samples and cases of RA significantly upregulated compared to OA. In contrast, in SM TFF3 protein was not significantly regulated. CONCLUSION: The data demonstrate the production of TFF3 in SM. Unexpectedly, SF contains all three known TFF peptides. As neither articular cartilage nor SM produce TFF1 and TFF2, we speculate that these originate with high probability from blood serum.

Differential Expression of TFF1 and TFF3 in Patients Suffering from Chronic Rhinosinusitis with Nasal Polyposis.

Trefoil family factor (TFF) proteins contribute to antimicrobial defense and the maintenance of sinonasal epithelial barrier integrity. Dysregulation of TFF expression may be involved in the development of chronic inflammation and tissue remodeling characteristically found in chronic rhinosinusitis with nasal polyposis (CRSwNP). Expressions of TFF1 and TFF3 were determined in specimens of middle nasal turbinate (MNT-0), bulla ethmoidalis (BE), and nasal polyps (NP) from CRSwNP patients (n = 29) and inferior nasal turbinate from a group of control patients (underwent nasal septoplasty, n = 25). An additional MNT sample was collected 6 months after functional endoscopic sinus surgery (FESS, MNT-6). TFF1 mRNA levels were significantly reduced in all specimens by approximately three- to five-fold, while TFF3 was increased in MNT-0, as compared with controls. Six months after surgery their levels were reversed to control values. CRSwNP patients with S. epidermidis isolated from sinus swabs showed upregulation of TFF3 in MNT and NP as compared with patients with sterile swabs. Target gene regulation was not affected by the presence of type 2 inflammation in patients with confirmed allergy. Results of this study imply participation of TFFs genes in the development of CRSwNP.

Trefoil Factor 3 Deficiency Affects Liver Lipid Metabolism.

BACKGROUND/AIMS: Tff3 protein plays a well recognized role in the protection of gastrointestinal mucosa. The role of Tff3 in the metabolism is a new aspect of its function. Tff3 is one of the most affected liver genes in early diabetes and fatty liver rodent models. The aim of this study was to investigate the effect of Tff3 deficiency on lipid and carbohydrate metabolism and on markers of oxidative stress that accompanies metabolic deregulation. METHODS: Specific markers of health status were determined in sera of Tff3 deficient mice, including glucose level, functional glucose and insulin tolerance. Composition of fatty acids (FAs) was determined in liver and blood serum by using gas chromatography. Oxidative stress parameters were determined: lipid peroxidation level via determination of lipid hydroperoxide and thiobarbituric acid reactive substances (TBARS), antioxidative capacity (FRAP) and specific antioxidative enzyme activity. The expression of several genes and proteins related to the metabolism of lipids, carbohydrates and oxidative stress (CAT, GPx1, SOD2, PPARα, PPARγ, PPARδ, HNF4α and SIRT1) was determined. RESULTS: Tff3 deficient mice showed better glucose utilization in the glucose and insulin test. Liver lipid metabolism is affected and increased formation of small lipid vesicles is noticed. Formation of lipid droplets is not accompanied by increased liver oxidative stress, although expression/activity of monitored enzymes is deregulated when compared with wild type mice. Tff3 deficient mice exhibit reduced expression of metabolism relevant SIRT1 and PPARγ genes. CONCLUSION: Tff3 deficiency affects the profile and accumulation of FAs in the liver, with no obvious oxidative stress increase, although expression/activity of monitored enzymes is changed as well as the level of SIRT1 and PPARγ protein. Considering the strong downregulation of liver Tff3 in diabetic/obese mice, presence in circulation and regulation by food/insulin, Tff3 is an interesting novel candidate in metabolism relevant conditions.

The predictive powers of plasma trefoil factor 3 or its related micro RNAs for patients with hepatocellular carcinoma.

BACKGROUND: Earlier diagnosis is beneficial for the prognosis of hepatocellular carcinoma (HCC). Alpha fetoprotein (AFP) is the most widely used biomarker for HCC, but its sensitivity and specificity are only 60 and 90%, respectively. Therefore, it is of great clinical significance to identify early prognostic biomarkers for HCC, especially a blood-based biomarker as it offers several advantages over tissue-based biomarkers. Trefoil factor 3 (TFF3), a novel secretory protein, was over-expressed in HCC tissues, indicating it might be a blood-based biomarker for HCC. In addition, circulating microRNAs have been investigated as biomarkers for HCC, indicating that miR-7-5p and miR-203a-3p, which are reported or predicted to target TFF3, also hold promise as blood-based biomarkers for HCC. METHODS: We enrolled 43 patients who were firstly diagnosed HCC and matched 47 control subjects without HCC. The levels of TFF3, miR-7-5p and miR-203a-3p were tested in the plasma of HCC patients. Moreover, we assayed the correlation of TFF3 with its related micro RNAs, miR-7-5p and miR-203a-3p, and evaluated their predictive powers for HCC. RESULTS: Decrease of TFF3 was associated with increase of miR-203a-3p in the plasma of HCC patients and they displayed potent predictive powers for HCC diagnosis. However, there was no significant change of plasma miR-7-5p between HCC and control group. CONCLUSION: Decrease of TFF3 correlated with increase of miR-203a-3p in the plasma of HCC patients and they could be additional biomarkers to improve sensitivity and specificity in the diagnosis of HCC.

Detection of HER2 amplification in formalin-fixed paraffin-embedded breast carcinoma tissue with digital PCR using two TFF3 sequences as internal reference.

PURPOSE: Droplet digital PCR (ddPCR) is a highly accurate method to determine DNA concentration and detect copy number variations. We developed an approach to assess HER2 gene amplification status using ddPCR with two sequences of TFF3 as reference probes. EXPERIMENTAL DESIGN: 76 templates of carcinoma DNA were prepared from formalin-fixed paraffin-embedded (FFPE) tissues. Digital PCR assay of the copy number of HER2 and TFF3 DNA was performed on the samples. The results were compared to prior fluorescent in-situ hybridation (FISH) assays performed on the same samples. RESULTS: The ddPCR assay had high concordance with the conventionally used immunohistochemistry (IHC) and FISH methods. The ddPCR method returned fewer indeterminate results than IHC. Concordance between a ddPCR plus FISH method and IHC plus FISH can rise to 98.7% (75/76) after validation is carried out. CONCLUSION: It's potentially possible to improve the sensitivity and specifity of HER2 ddPCR assays using reference sequences not co-localized with HER2 on chromosome 17, and combining results from multiple sequences. Adopting an approach based on ddPCR HER2 assays plus FISH could lead to reduced costs, labour, and time consumption compared to current IHC plus FISH standard, while not losing precision.

Lentivirus-mediated shRNA interference of trefoil factor 3 blocks cell viability, migration and invasion in the papillary thyroid carcinoma cells.

Trefoil factor 3 (TFF3), a regulatory protein composed of 59 amino acids, has been suggested to be involved in pathogen- esis, proliferation, invasion, migration and apoptosis in multiple malignant tumors. However, the roles of TFF3 concerning the viability, migration and invasion in papillary thyroid carcinoma cells have not yet been studied. This study aimed to investigate the effect of TFF3 knockdown on a thyroid papillary carcinoma TPC-1 cell line both in vitro and in vivo. In the present study, lentivirus-mediated short hairpin RNA (shRNA) targeting TFF3 plasmids were first constructed and stable TPC-1 cells were obtained while their TFF3 gene was silenced with either shTFF3-TPC-1, or a scrambled shRNA control. TFF3 expression was detected using quantitative real-time PCR and western blot analyses. The TPC-1 cell viability was measured by CCK-8 assay and colony formation. The cell migration and invasion were assessed by wound scratch assay and transwell filters. AKT phosphorylation, MMP-9, and BCL-2 expression levels were detected by western blot analyses. Our results showed that TFF3 knockdown significantly inhibits TPC-1 cell viability, migration and invasion. AKT phosphoryla- tion, MMP-9, and BCL-2 levels were all remarkably depressed in TFF3 knockdown TPC-1 cells. Using a thyroid papillary carcinoma xenograft mouse model, we further investigated the effects of TFF3 knockdown in vivo. Significantly delayed xenograft emerging, slower growth rate and lower final tumor weights and volumes were observed in the shTFF3 group as compared to the control group. As expected, the expression levels of MMP-9 and BCL-2 in the xenograft are consistent with those of shTFF3-TPC-1 and shTFF3-TPC-1 cells in vitro. Our results suggest that TFF3 plays a vital role in the viability and oncogenesis of TPC-1 cells and may be a potential target for effective treatment of thyroid papillary carcinoma.