In vivo results revealed that IQGAP1 promoted tumor growth and metastasis. Co-immunoprecipitation studies demonstrated that IQGAP1 interacted with both DVL2 and β-catenin. Moreover, knockdown of DVL2 reversed IQGAP1-induced EMT. Our findings thus confirmed that IQGAP1 could be used as a potential target for PDAC treatment.IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that participates in several cellular functions, including cytoskeletal regulation, cell adhesion, gene transcription and cell polarization. IQGAP1 has been implicated in the tumorigenesis and progression of several human cancers. However, the role of IQGAP1 in pancreatic ductal adenocarcinoma (PDAC) is still unknown. We found that IQGAP1 expression was an independent prognostic factor for PDAC. IQGAP1 upregulation significantly promoted cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT), whereas IQGAP1 downregulation impaired its oncogenic functions. Overexpression of IQGAP1 increased the protein level of Dishevelled2 (DVL2) and enhanced canonical Wnt signaling as evidenced by increased DVL2 level, β-catenin transcriptional activity, β-catenin nuclear translocation and expression of the direct target genes of β-catenin (cyclin D1 and c-myc). In contrast, knockdown of IQGAP1 decreased the level of DVL2 and attenuated Wnt/β-catenin signaling.  Despite progress in treatment strategies, the decrease in mortality rate for this disease is still slow2 due to aggressive growth, distant metastasis, chemoresistance and difficulties in early diagnosis5. Pancreatic ductal adenocarcinoma (PDAC) accounts for more than 95% of all PC cases6. Therefore, there is an urgent need to explore diagnostic molecular biomarkers and targets with high therapeutic efficacy to improve the prognosis of PDAC patients.Pancreatic cancer (PC) remains one of the most lethal malignancies with a 5-year survival rate of less than 5%7. Several cellular functions are regulated by the multidomain structures of IQGAP1 through its binding with many other partners, which is mainly mediated by the RGCT or IQ regions9. For example, IQGAP1 functions as a scaffold in the MEK/ERK cascade by binding to MEK1/2, ERK1/2 and B-Raf via its IQ region10. Accumulating evidence indicates that IQGAP1 is an oncogene12. IQGAP1 expression has been reported to be upregulated in several types of human cancer tissues and cell lines, such as hepatocellular carcinoma12, thyroid cancer13, breast cancer14, esophageal squamous cell carcinoma15, non-small cell lung cancer16 and PC17.As one of the evolutionally conserved scaffold protein family members, IQ motif-containing GTPase-activating protein 1 (IQGAP1) includes five domains, namely, the calponin homology domain interacting with F-actin, GAP-related domain interacting with small GTPases (Rac1 and Cdc42), poly-proline protein-protein domain interacting with ERK, IQ domain interacting with four IQ motifs and RasGAP C-terminusin vivo and in vitro models. Finally, we explored the effect of IQGAP1 on the Wnt/β-catenin signaling pathway in PDAC cells.However, the function and underlying mechanism of IQGAP1 in PDAC have not been fully evaluated. The present study aimed to investigate the prognostic values and biological functions of IQGAP1 and the associated molecular mechanisms in PDAC. We first evaluated the expression level of IQGAP1 in PDAC tissues using immunohistochemistry (IHC) and investigated the association of IQGAP1 expression level with clinicopathological features and patient survival. Then, we studied the role of IQGAP1 in the progression of PDAC by using both L)  ≤4) containing 30 samples and the high-IQGAP1 group (IQGAP1H)  containing 57 samples. The percentage of high expression of IQGAP1  in PDAC tissues was significantly higher than that  in matched peritumor tissues  rates than patients in the IQGAP1L group  and 33.0 months (95% CI 0.085–65.915), respectively. Univariate analysis indicated that the following variables were significantly related to OS: histological differentiation (P = 0.047), N stage (P < 0.001), and IQGAP1 expression (P = 0.004). In multivariate analysis using Cox proportional hazards models, N1 stage , poor tumor differentiation , and high IQGAP1 expression  were correlated with OS  were smaller than those of tumors generated from negative control cells (SW1990-shNC)  Fig.  (mean voNC) Fig. . AdditioNC) Fig. . Therefoin vitro.To further assess the oncogenic role of IQGAP1, we next overexpressed IQGAP1 in the PC cell line PANC-1 (PANC-1-IQGAP1 cells) by transiently transfecting PANC-1 cells with IQGAP1 plasmids. Western blotting and qRT-PCR analysis confirmed a significant increase in IQGAP1 expression in PANC-1-IQGAP1 cells relative to that in control cells Fig. . Overexp18, but the correlation between IQGAP1 and DVL2 in PC cell lines have not been studied. We thus examined whether endogenous IQGAP1 interacts with DVL2 and components of the Wnt/β-catenin pathway. Co-IP experiments verified that IQGAP1 and DVL2 interacted with each other in HEK-293T, SW1990 and CFPAC-1 cells 26 and promote RAS-MAP kinase-driven cancer invasion27. In addition, a significant increase in angiogenesis by the overexpression of IQGAP1 was found in an in vivo mouse model of breast cancer28. Moreover, IQGAP1 SUMOylation can stabilize IQGAP1 by reducing protein ubiquitination and thus promote the development of colorectal cancer29. It has been reported that IQGAP1 interacts with K-Ras and increases ERK1/2 phosphorylation to modulate K-Ras pathway in PC30. Although Jin et al. have demonstrated that IQGAP1 expression is positively associated with the degree of metastasis of PC in vitro31, to fully elucidate the possible functional significance of IQGAP1 in PC cell lines, we performed both gain- and loss-of-function assays. We observed that the knockdown of IQGAP1 in the two cell lines SW1990 and CFPAC-1 not only inhibited cell proliferation in vitro but also suppressed tumor growth and metastasis in vivo. Conversely, the upregulation of IQGAP1 enhanced proliferation and migration.IQGAP1 is a scaffold protein that interacts with distinct proteins to regulate cancer cell proliferation and migration19, and IQGAP1 overexpression promotes the transcription and translocation of β-catenin into the nucleus in hepatocellular carcinoma32. In contrast, in human colon adenocarcinoma, IQGAP1 knockdown via siRNA cannot alter the expression levels and localization of β-catenin20. Our results showed that IQGAP1 knockdown can inhibit canonical Wnt signaling in PC cells as evidenced by decreased levels of both cytoplasmic and nuclear β-catenin, as well as the downstream target genes of β-catenin including cyclin D1 and c-Myc, consistent with previous reports showing that IQGAP1 can regulate β-catenin nuclear localization, which is involved in cell transformation via the Wnt signaling pathway19. Our results indicated that the aberrant activation of the canonical Wnt signaling pathway is induced by IQGAP1 upregulation. These results indicate that IQGAP1 is critical for the progression of PC and plays an important role in the aberrant activation of the canonical Wnt/β-catenin pathway.Depletion of IQGAP1 can decrease the Wnt-induced nuclear accumulation of β-catenin during early embryogenesis33. DVL has three homologues . Some DVL2-associated oncoproteins, such as Forkhead box (FOX) transcription factors (FOXK1 and FOXK2)34, ataxia-telangiectasia group D complementing gene (ATDC)35, and RAP1B36, translocate DVL2 into the nucleus and therefore activate the Wnt/β-catenin signaling pathway. However, some negative regulators such as DVL-DEP domain-Interacting Protein (DDIP) destroy the TCF4/β-catenin complex37 and MLLT4 (AF6) independent of the canonical Wnt/β-catenin signaling pathway38 and ultimately inhibit cell proliferation. DVL2 has been demonstrated to contribute to PC tumorigenesis by inducing the stabilization of β-catenin35. Consistent with the result of other studies, our results revealed that DVL2 can interact with IQGAP1 and that knockdown of DVL2 in PANC-1 cells overexpressing IQGAP1 can reverse the increase in β-catenin levels. By co-IP assays, we confirmed that IQGAP1 forms a complex with DVL2 and β-catenin and may mediate the interaction between DVL2 and β-catenin. We found that IQGAP1 can promote DVL2 nuclear translocation and regulate the protein level but not the mRNA level of DVL2, suggesting that changes in DVL2 stability play an important role in regulating the Wnt/β-catenin signaling pathway. However, the detailed mechanisms remain to be elucidated.DVL is critical for protecting β-catenin from degradation in canonical Wnt signaling, where the interaction of DVL with both c-Jun and β-catenin promotes Wnt signaling-stimulated transcription39. IQGAP1 has been documented to be tightly linked with the Wnt/β-catenin signaling pathway32 and to be involved in regulation of cell proliferation and EMT40. DVL2 is responsible for the regulation of EMT in MCF-7 cells41. However, the role of IQGAP1 and DVL2 in the regulation of EMT mediated by Wnt/β-catenin signaling remains obscure. In our study, we provided evidence that IQGAP1 augmented the expression of mesenchymal markers (N-cadherin and Vimentin) and EMT-activating transcription factors  while decreasing the expression of the epithelial marker E-cadherin. We also demonstrated that the knockdown of DVL2 inhibited EMT induced by IQGAP1 overexpression. Therefore, we concluded that IQGAP1 may serve an important mediator of EMT through DVL2.The loss of tumor cell-cell adhesion and gain of tumor invasion enabled by EMT initiates tumor metastasis. E-cadherin-mediated cell-cell adhesion has been proven to be reduced by the overexpression of IQGAP1 via direct interaction with CDC42 and Rac1in vitro and in vivo results extend our understanding of the underlying molecular mechanism of IQGAP1 in the regulation of EMT and cell proliferation mediated by Wnt/β-catenin signaling. To better reveal the intrinsic mechanism of IQGAP1-induced carcinogenesis, larger populations of patients, longer follow-up and further molecular studies in PC are still needed to support our findings.In conclusion, this study indicated that the overexpression of IQGAP1 is associated with unfavorable clinicopathological features and poor survival in patients with PDAC. IQGAP1 can also promote pancreatic tumorigenesis and progression, making it an attractive therapeutic target or prognostic molecular biomarker of PDAC. Our findings show that IQGAP1 regulates Wnt/β-catenin signaling by interacting with DVL2 and β-catenin. Both th edition of International Union Against Cancer guidelines. Approval from ethics committee of Lianyungang Clinical College of Nanjing Medical University and informed consent from each patient were obtained before enrollment into the study. This study was performed in accordance with the Declaration of Helsinki.Human PDAC samples and peritumor samples were obtained from the Department of Hepatobiliary Surgery, Lianyungang Clinical College of Nanjing Medical University. The patients were histopathologically diagnosed with primary PDAC and had not received any anticancer therapy before surgery. PDAC tumors were staged according to the 7Based on the silencing effect of small interfering RNAs (siRNAs) against IQGAP1, we chose sense 5′-GCGACAAAGUCCUGAACAU-3′ and antisense 5′-AUGUUCAGGACUUUGUCGC-3′ to be inserted into the lentiviral expression vector hU6-MCS-Ubiquitin-EGFP-IRES-puromycin . GFP in all lentiviral vectors was used to monitor transfection efficiency. SW1990 cells were seeded in 6-well plates and transfected with IQGAP1-targeting shRNA and negative control shRNA. Forty-eight hours after infection, the cells were treated with puromycin (1 μg/ml) for approximately two weeks to generate stable transfected cells.6) into the mice (6 mice per group). The mice were randomly divided into two groups after 24 days of tumor implantation. Tumor volume was calculated by the following formula: volume = (length × width2)/2. For metastasis study, SW1990 cells (1 × 106) in 50 μL phosphate-buffered saline were orthotopically injected into the major pancreatic duct (8 mice per group). Three months after injection, the mice were euthanized, and suspected metastatic tissues were subjected to hematoxylin and eosin (H&E) staining for the pathologic examination of metastatic nodules.All male BALB/c nude mice, aged 4–6 weeks, used in this study were purchased from Model Animal Research Center at Nanjing University . All the animal protocols were reviewed and approved by the Animal Care Committee of Nanjing University in accordance with the guidelines of the Institutional Animal Care and Use Committee. To assess tumor growth, we subcutaneously implanted SW1990 cells  construction, immunohistochemistry and scoring, small interfering RNA and plasmid vector-related assays, quantitative real-time reverse transcription polymerase chain reaction, western blotting, CCK-8 assay, colony formation assay, wound healing assay, transwell migration assay, co-immunoprecipitation (co-IP) experiments, and immunofluorescence imaging, please see Supplemental File.P value < 0.05 was considered statistically significant.Associations of categorical data between IQGAP1 expression and clinicopathological features were assessed using the chi-square test and Fisher’s exact test. Possible differences in overall survival (OS) were examined by the Kaplan-Meier analysis and log-rank test. Prognostic factors associated with OS in univariate analysis were evaluated using Cox regression models for multivariate analysis. Student’s t-tests were used to determine the differences between two independent groups, and the results were expressed as the mean ± SD. All statistical analyses were performed using SPSS 23 statistical software . Supplemental Information


An altered Wnt-signaling activation has been reported during Barrett’s esophagus progression, but with rarely detected mutations in APC and β-catenin (CTNNB1) genes.In this study, a robust in-depth expression pattern analysis of frizzled receptors, co-receptors, the Wnt-ligands Wnt3a and Wnt5a, the Wnt-signaling downstream targets Axin2, and CyclinD1, as well as the activation of the intracellular signaling kinases Akt and GSK3β was performed in an in vitro cell culture model of Barrett’s esophagus. Representing the Barrett’s sequence, we used normal esophageal squamous epithelium , metaplasia (CP-A) and dysplasia (CP-B) to esophageal adenocarcinoma (EAC) cell lines  and primary specimens of squamous epithelium, metaplasia and EAC.A loss of Wnt3a expression was observed beginning from the metaplastic cell line CP-A towards dysplasia (CP-B) and EAC (OE33 and OE19), confirmed by a lower staining index of WNT3A in Barrett’s metaplasia and EAC, than in squamous epithelium specimens. Frizzled 1–10 expression analysis revealed a distinct expression pattern, showing the highest expression for Fzd2, Fzd3, Fzd4, Fzd5, Fzd7, and the co-receptor LRP5/6 in EAC cells, while Fzd3 and Fzd7 were rarely expressed in primary specimens from squamous epithelium.Despite the absence of an in-depth characterization of Wnt-signaling-associated receptors in Barrett’s esophagus, by showing variations of the Fzd- and co-receptor profiles, we provide evidence to have a significant role during Barrett’s progression and the underlying pathological mechanisms.The online version of this article (10.1186/s12876-019-0957-5) contains supplementary material, which is available to authorized users. Esophageal cancer is often associated with a poor overall survival when diagnosed in advanced stages . SurgeryIn contrast to the slowly decreasing incidence of squamous cell carcinoma, the incidence of esophageal adenocarcinoma (EAC) has highly increased over the past decades , 4.Mostly, EAC develops from a long history of Barrett’s esophagus, which results from long-lasting gastroesophageal reflux disease (GERD). Barrett’s metaplasia is defined as a condition, where the squamous epithelium of the esophagus is replaced by columnar epithelium with goblet cells , 6. The The Wnt−/β-catenin signaling pathway is responsible for cell growth, motility and differentiation during embryogenesis . In mammIn different cancer entities, the Wnt−/β-catenin signaling pathway seems to play a key role during carcinogenesis. In colorectal cancer, a mutation of the APC gene is often observed . In addiHepatocellular carcinoma (HCC), hepatoblastoma and hepatocellular adenoma often show mutations of β-catenin . OverallAn activated Wnt−/β-catenin signaling pathway could also be a potential mechanism for the progression of EAC. Clément et al. observed a methylation of the APC promotor, which goes along with a lack of APC expression in Barrett’s esophagus and EAC. In addition to that, a promotor methylation of the Wnt antagonist secreted frizzled receptor protein 1 (SFRP1), which is associated with a loss of function of SFRP1, was found in Barrett’s esophagus and EAC more often than in normal squamous mucosa . Cell cuOverall, these findings strongly indicate an activation of the Wnt−/β-catenin signaling pathway during EAC development. In this study, we aimed to investigate the role of Wnt−/β-catenin signaling pathway activation in different stages during the progression from squamous epithelium to EAC in vitro. Analyzing the expression of Wnt3a, activating the canonical, and Wnt5a, the non-canonical Wnt−/β-catenin signaling pathway, membranous Fzd-receptors, intracellular molecules and downstream targets, we want to give a brief overview of Wnt-pathway genes and a comprehensive understanding of the molecular background for Wnt-signaling in EAC progression in an in vitro Barrett’s cell culture model. Nevertheless, we provide exemplary insights in the presence of Wnt-molecules and -receptors in specimens from patients with Barrett’s esophagus, high-grade intraepithelial neoplasia, and esophageal adenocarcinoma.later®  for a maximum of 24 h and stored at - 80 °C until further usage. Seven specimens of squamous epithelium, Barrett’s mucosa, HGIN, and EAC were investigated. The diagnoses of Barrett’s mucosa, HGIN, or EAC were confirmed by an expertized GI pathologist.For expression analysis of the Wnt-pathway components, endoscopic biopsies were taken from Barrett’s mucosa with intestinal metaplasia, high-grade intraepithelial neoplasia, esophageal adenocarcinoma. Normal mucosa was taken from patients with Barrett’s mucosa using a safety distance of at least 3 cm to the Barrett’s mucosa. Biopsies were placed directly to RNARecombinant human Wnt3a  was purchased from R&D Systems . The following antibodies phospho-GSK3β-Ser9 (D85E12), GSK3β (D5C5Z), phospho-Akt-Ser473 (D9E), Akt (C67E7), phospho-β-catenin-Ser552 (D8E11), and β-catenin (D10A8) were all purchased from Cell Signaling Technology  and the β-actin antibody from Sigma-Aldrich . The peroxidase-conjugated goat anti-rabbit and goat anti-mouse antibodies were purchased from Jackson ImmunoResearch .2.In total, six different cell lines, representing Barrett’s sequence, were cultivated. EPC1-hTERT (EPC-1) and EPC2-hTERT (EPC-2) were a generous gift from Dr. Hiroshi Nakagawa . They ar2. After one day, the medium was changed and one day later, cells were incubated with FCS free medium for three hours. The cells were stimulated with 200 ng rhWnt3a at 37 °C, 5% CO2 for one hour. Removing medium and washing two times with cold PBS, the stimulation with Wnt3a was stopped.EPC-1, EPC-2, CP-A, CP-B, OE33 and OE19 cells with a confluence of about 80% were seeded with 250,000 cells per well in a 6-well plate and incubated at 37 °C with 5% COFor RNA isolation from cell cultures, the RNeasy Plus Mini Kit  was used. RNA isolation from biopsies was done with TRI Reagent® , followed by DNase  treatment. CDNAs were synthesized from 250 ng to 1000 ng of total RNA using the RevertAid RT synthesis kit  according to the manufacturer’s protocol.For isolating RNA from paraffin embedded biopsies, we used the AllPrep DNA/RNA FFPE Kit  according to the manufacturer’s protocol. 125 ng of total RNA were used to synthesize cDNA using SuperScript IV Reverse Transcriptase .Quantitative RT-PCR was conducted by using Takyon No ROX SYBR®Core Kit dTTP Blue  with 1 μl of cDNA and 14 μl of reaction mixture. Primers used for quantitative RT PCR are shown in Table Using DreamTaq Green PCR Master Mix , polymerase chain reaction was performed with 1 μl of cDNA per 20 μl reaction mix. The following protocol was used for 40 cycles: denaturation 98 °C for 2 min, annealing 94 °C for 10 s, elongation 60 °C for 20 s. 10 μl of cycled PCR products were applied to 2% ethidiumbromid stained agarose gel and visualized with UV light. Primers used for qualitative RT PCR are shown in Table Harvested cells were lysed in RIPA buffer. Using the method of Bradford, protein concentrations were detected. 20 μg of protein were separated on 8% or 10% sodium dodecyl sulphate (SDS)-polyacrylamide gels and blotted on nitrocellulose membranes. After blocking the membranes with 5% low fat milk for one hour, they were incubated with a specific primary antibody over night at 4 °C. Detecting bound antibody, a peroxidase conjugated secondary antibody goat anti-mouse or goat anti-rabbit was used incubating for 1 h at room temperature. Protein bands were visualized with ECL chemiluminescence detection . The dilutions of the different antibodies are shown in Table 2O2 for 20 min at 4 °C. The slices were cooked in citrate buffer with a pressure cooker for 10 min. Blocking unspecific bindings was performed in several steps, first with 5% NormalGoatSerum  for 20 min at room temperature and second with Reagents from the Streptavidin/Biotin blocking kit , as described in the manufacturer’s protocol. Diluted 1:50 in 1% BSA in PBS, the monoclonal primary antibody against WNT3A  was incubated over night at 4 °C. Detecting bound antibody, a biotin-SP conjugated goat anti-mouse  and a peroxidase conjugated Streptavidin , both diluted 1:1000 in 1% BSA in PBS, were utilized for 1 h at room temperature. Results were visualized with diaminobenzidine and counterstained with hematoxylin. Breast cancer tissue was used as positive control. Evaluation was done using a staining index  with categorization for analyzing the stroma, the squamous epithelium, and the metaplastic or EAC tissue.Paraffin embedded biopsies from squamous epithelium, Barrett’s metaplasia, HGIN and EAC were cut in 3 μm sections and deparaffinized using a series of different concentrated alcohols and xylol. Endogenous peroxidase was blocked by 3% Hp-value of less than 0.05 was considered statistically significant.Analyses were performed using GraphPad Prism 6 . Results are presented as mean ± standard error (S.E.M.) and a Along Barrett’s sequence, Wnt3a and Wnt5a were expressed differently. Significantly elevated Wnt3a expression levels were detected in squamous cell lines EPC-1 and EPC-2, while metaplastic (CP-A), dysplastic (CP-B) and carcinoma cell lines  showed only marginal expression of Wnt3a. Wnt5a was expressed in EPC-1 and EPC-2. However, the dysplastic cell line CP-B demonstrated the highest expression levels of Wnt5a. In contrast, the metaplastic cell line CP-A and the carcinoma cell lines OE33 and OE19 did not express Wnt5a  and a moderate expression of Wnt5a. However, 4 of 7 specimens of Barrett’s mucosa, 1 of 7 specimens of HGIN, and 2 of 7 specimens from EAC displayed a Wnt3a expression  displayed differences between the squamous cell lines EPC-1 and EPC-2, with significant higher levels of pβ-catenin in EPC-2. Overall, EPC-1, CP-A, CP-B, and OE 33 showed low levels of pβ-catenin. The presence of pβ-catenin could not be detected in OE19.We revealed elevated expression levels of pGSK3β (Ser9) in the squamous cell lines EPC-1 and EPC-2, as well as in the carcinoma cell line OE19. In contrast, OE33 showed a low expression of pGSK3β. The metaplastic cell line CP-A expressed balanced levels of pGSK3β and GSK3β. Analysis of the kinase Akt showed different protein expression levels of phosphorylated Akt Ser473) in the different cell lines. The cell lines EPC-1, EPC-2 and CP-A expressed only marginal levels of pAkt. Balanced expression of pAkt and Akt was found in CP-B and OE19. We detected a loss of pAkt expression in the carcinoma cell line OE33  for 1 h, we analyzed the expression of the downstream targets Axin2 and Cyclin D1. Only OE33 showed significant increased levels of Axin2 expression levels compared to controls. All other cell lines investigated did not show any alterations of Axin2 expression after Wnt3a treatment Fig. b.Furthermore, decreased expression levels of Cyclin D1 were found in the squamous , metaplastic (CP-A) and dysplastic (CP-B) cell lines after stimulation with Wnt3a. The cell line OE33 expressed increased levels of CyclinD1, while OE19 responded marginally Fig. c. WesterExpression levels of LRP5/6 and Axin2 were analyzed in patient-derived biopsies. In concordance with results presented above, we found elevated expression levels of LRP5/6 in EAC as compared to metaplasia specimens. Additionally, the expression of Axin2 was significantly higher in EAC specimens as well  compared to intestinal metaplasia, HGIN and adenocarcinoma cells Fig. a. The amIn this work, we provide an in-depth characterization of the Wnt-signaling pathway components on a cellular level using both, an in vitro cell culture model of Barrett’s esophagus and evidence from human data to elucidate changes in receptor, ligand, intracellular signaling cascade and downstream target of the Wnt-signaling pathway. Aberrations in Wnt-signaling had been observed in many malignancies, including colon cancer , hepatocCharacterizing Wnt-signaling pathway components in an in vitro Barrett’s esophagus cell culture model was carried out in squamous epithelium (EPC-1 and EPC-2), metaplastic (CP-A), high-grade dysplastic (CP-B) and in adenocarcinoma (OE33 and OE19) cells. We could show a wide spread of frizzled receptor expression throughout Barrett’s sequence, demonstrating mostly an increase in frizzled receptor expression in the more advanced Barrett’s cell lines for Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, and Fzd7 Fig. . In vitrSince the Wnt-signaling pathway has been reported to be activated in the progression of Barrett’s esophagus, a robust characterization of its receptors is still missing , 17, 26.A potential alternative pathway, which might be involved in the Wnt-signaling pathway activation along the progression of Barrett’s esophagus, is the NF-κB-pathway, triggered by an increased TNF-α-receptivity in the more advanced stages of Barrett’s esophagus –34. AcidWe demonstrated for the first time a comprehensive analysis of all 10 frizzled receptors and their co-receptors LRP5 and LRP6 in an in vitro cell culture model of Barrett’s esophagus and in primary human specimens from normal squamous epithelium, Barrett’s mucosa, HGIN, and EAC.The in depth characterization of the Wnt-receptor and Wnt-ligand expression pattern will allow new insights into the activation of the Wnt-signaling pathway in Barrett’s esophagus and Barrett’s carcinoma, as well as in the pathomechanisms involved in the progression of Barrett’s esophagus. A concluding answer of the exact molecular level of Wnt-axis activation needs further elucidation and an exact molecular profile of both, a useful in vitro cell culture model as well as in vivo analyses of Barrett’s patients’ specimens.Additional file 1:Figure S1. Phosphoryation of Akt, GSK3β and β-catenin after Wnt3a treatment in EPC1, EPC2, CP-A, CP-B, OE33 and OE19 cells. Representative Westernblots related to the analysis shown in Fig.


Bone defects caused heavy social and economic burdens worldwide. Nel‐like molecule, type 1 (NELL‐1) could enhance the osteogenesis and the repairment of bone defects, while the specific mechanism remains to be elucidated. Circular RNAs (circRNAs) have been found to play critical roles in the tissue development and serve as biomarkers for various diseases. However, it remains unclear that the expression patterns of circRNAs and the roles of them played in recombinant NELL‐1‐induced osteogenesis of human adipose‐derived stem cells (hASCs). In this study, we performed RNA‐sequencing to investigate the expression profiles of circRNAs in recombinant NELL‐1‐induced osteogenic differentiation and identified two key circRNAs, namely circRFWD2 and circINO80. These two circRNAs were confirmed to be up‐regulated during recombinant NELL‐1‐induced osteogenesis, and knockdown of them affected the positive effect of NELL‐1 on osteogenesis. CircRFWD2 and circINO80 could interact with hsa‐miR‐6817‐5p, which could inhibit the osteogenesis. Silencing hsa‐miR‐6817‐5p could partially reverse the negative effect of si‐circRFWD2 and si‐circINO80 on the osteogenesis. Therefore, circRFWD2 and circINO80 could regulate the expression of hsa‐miR‐6817‐5p and influence the recombinant NELL‐1‐induced osteogenic differentiation of hASCs. It opens a new window to better understanding the effects of NELL‐1 on the osteogenic differentiation of hASCs and provides potential molecular targets and novel methods for bone regeneration efficiently and safely. Unlike linear RNAs, they hold a covalently closed loop, keeping them stable and resistant to RNase R digestion.In present study, we performed RNA‐sequencing and compared the expression profiles of circRNAs between recombinant NELL‐1‐induced osteogenic differentiation group (NG) and general osteogenic differentiation group (OG). The potential genomic function was investigated by Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Two key circRNAs, namely circRFWD2 and circINO80, were identified to be up‐regulated during recombinant NELL‐1‐induced osteogenesis. Our results indicated that circRFWD2 and circINO80 could regulate the expression of hsa‐miR‐6817‐5p and influence the recombinant NELL‐1‐induced osteogenesis of hASCs.22.1The hASCs were purchased from Cyagen company. All the hASCs were separated into two groups: recombinant NELL‐1‐induced osteogenesis group (NG) and general osteogenesis group (OG).2 at 37℃.The OriCell™ general medium, which was utilized to culture hASCs, contained 10% mesenchymal stem cell‐qualified foetal bovine serum (FBS), 1% glutamine and 1% penicillin‐streptomycin. The osteogenic induction was initiated once the confluence of hASCs reached 80%. The general osteogenic inductive medium included basic medium, 10 nM dexamethasone, 10 mM β‐glycerophosphate and 50 μg/mL vitamin C (Sigma‐Aldrich), which was applied to culture the hASCs in OG. The hASCs in NG was culture in the general osteogenic inductive medium supplemented with 300 ng/mL of recombinant human NELL‐1 protein (R&D Systems).2.2Alizarin red s staining was performed to detect the mineral deposition. Based on the protocol, the samples were fixed in 4% paraformaldehyde solution at 4°C for 20 minutes. After washed three times with distilled water, the samples were stained with 0.1% ARS for 20 minutes at 25℃.2.3Alkaline phosphatase staining was implemented with the Leukocyte Alkaline Phosphatase Kit 86C (Sigma) on the basis of the manufacturer's instruction. In brief, samples were washed with PBS and fixed in citrate solution for 30 seconds. Then, they were stained with the solution of FRV alkaline, naphthol AS‐BI alkaline and sodium nitrite for 15 minutes, protected from light during this process.The Alkaline Phosphatase Assay Kit (Beyotime) was used for the analysis of ALP activity. The samples were cracked with RIPA lysis buffer, and supernatant was transferred to a 96‐well plate. The 96‐well plate was incubated at 37℃ for 30 minutes after p‐nitrophenol, and the reaction substrates were added. Finally, the ALP activity was calculated using p‐nitrophenyl substrate at 405 nm.2.4The total RNAs were isolated from NG and OG with the Trizol reagent (Invitrogen). The RNA purity and concentration of samples were determined by NanoDrop ND‐1000 (NanoDrop Thermo). The RNA integrity of samples was detected by denaturing agarose gel electrophoresis. In general, 3 units/μg of RNase R  were applied to treat 5μg RNAs for 15 minutes at 37°C to remove linear RNA. The rRNAs of the Rnase R–treated RNAs were depleted using Ribo‐Zero Magnetic Gold Kit .2.5CircRNA sequencing and RNA library construction were performed by CloudSeq Biotech Inc. The RNA libraries were constructed with a TruSeq Stranded Total RNA Library Prep Kit (Illumina). The quality and quantity of RNAs in the libraries were determined by the BioAnalyzer 2100 system (Agilent Technologies). RNA‐Seq sequencing was conducted on the Illumina HiSeq platform. The Illumina HiSeq 4000 sequencer was used to acquire paired‐end reads.P values < .05, were considered as significant differential expression.Edger software was utilized to identify the differentially expressed circRNAs among NG and OG.The GO and KEGG analyses were used to predict the functions of differentially expressed circRNA‐associated genes. GO analysis measured biological processes, cellular components and molecular functions. KEGG pathway analysis was used to identify pathways related to the target mRNAs of circRNAs. To investigate the potential functions of the differentially expressed circRNAs, the prediction software was utilized to show the interactions of these circRNAs with the targeted miRNAs. The prediction of miRNA‐binding sites of the targeted mRNAs was performed on the basis of TargetScanHuman 7.2 and miRanda.2.6The mimic and the inhibitor of hsa‐miR‐6817‐5p, miRNA control, circRFWD2 siRNA, circINO80 siRNA and control vector were synthesized by GenePharma Co. and shown in Table 2.7For the selected circRNAs, total RNAs (3 μg) were employed for first strand cDNA synthesis with dNTP Mix (HyTestLtd), RNase inhibitor (Enzymatics) and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). The qRT‐PCR was performed on an Applied Biosystems 7500 Fast Real‐Time PCR System (Applied Biosystems) using SYBR Green master mix (Cloudseq). The primers of circRNAs and genes were synthesized by Sangon and shown in Table −ΔΔCt.The cDNA synthesis and quantitative detection of miRNAs were performed with the miRNA qRT‐PCR Detection Kit (GeneCopoeia). The primer of hsa‐miR‐6817‐5p was designed by GeneCopoeia. U6 was used for normalization. The relative expression was calculated by the formula 22.8The protein levels of RUNX2 and bone sialoprotein (BSP) were determined by Western blot. Radioimmunoprecipitation assay (RIPA) lysis buffer was used to extract total cell protein. Protein concentration was determined by the BCA Protein Assay Kit (Thermo). Equal microlitres of protein samples were loaded onto sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and then, they were transferred onto PVDF membranes (Millipore). The PVDF membranes were incubated with monoclonal antibodies against anti‐RUNX2 , anti‐BSP  and GAPDH  overnight at 4°C. After washed with TBST, the membranes were incubated with corresponding secondary antibodies  for 2 hours. The band intensity was determined by ImageJ software. All the target bands were normalized to GAPDH band.2.9P‐value < .05 was considered as statistical significance.Quantitative data were expressed as means ± standard deviation (SD), and all the experiments were performed three times at least. The statistical analysis was performed with SPSS 17.0 software. The differences between two groups were analysed by unpaired t test, while one‐way analysis of variance (ANOVA) were utilized to identify the differences between more than two groups. 33.1P < .01) . Meanwhile, the expression levels of osteogenic markers, namely COL I and RUNX2, were up‐regulated significantly in NG   and decreased in inhibitor group significantly (P < .05) , and those circRNAs were up‐regulated in hsa‐miR‐6817‐5p inhibitor group without significance  (Figure After induced for 7 days, the expression level of hsa‐miR‐6817‐5p was lower significantly in NG than that in OG (4CircRNAs are highly conserved among species due to the closed loop structures,It is reported that circRNAs could interact with RNA‐binding protein and affect the protein function.In this study, we performed GO analysis to annotate biological functions of parental genes of differentially expressed circRNAs. The biological processes, in which most circRNAs were enriched, were the regulation of cell cycle process, the organelle organization and the mitotic nuclear division. The most enriched cellular components terms were the intracellular part, the nucleoplasm and the intracellular. For molecular function aspect, the most enriched terms were related to the protein binding, the guanyl‐nucleotide exchange factor activity and the small GTPase binding. The KEGG pathway analysis suggested most of the differentially expressed circRNAs were enriched in the Glutamatergic synapse, THs signalling pathway and p53 signalling pathway. p53 signalling pathway was reported to regulate bone formation negatively in vivo and in vitro, under the control of BMPs and IGFs pathways.Additionally, circRFWD2 and circINO80 were identified to be up‐regulated during recombinant NELL‐1‐induced osteogenesis by RNA‐seq, which was validated by qRT‐PCR. When inhibiting circRFWD2 and circINO80, respectively, we found the osteogenic markers, RUNX2 and OPN, were down‐regulated obviously. The ALP activity and calcium nodules staining were further confirmed the results above. During recombinant NELL‐1‐induced osteogenesis, the expression of RUNX2 and OPN were also decreased significantly in both of circRFWD2 and circINO80 knockdown groups than group NC. Therefore, it is suggested that these two circRNAs might influence recombinant NELL‐1‐induced osteogenesis positively.Our study showed that hsa‐miR‐6817‐5p was down‐regulated during the recombinant NELL‐1‐induced osteogenesis, and it could inhibit the recombinant NELL‐1‐induced osteogenic differentiation. It is suggested that hsa‐miR‐6817‐5p was closely associated with hsa_circ_0061012, which was up‐regulated in psoriasis significantly. Psoriasis was related well to the regulation of NF‐kappaB,Moreover, we found that the binding sites of hsa‐miR‐6817‐5p could match the sequences of circRFWD2 and circINO80, respectively, and the expression levels of these two circRNAs were negatively correlated with hsa‐miR‐6817‐5p. Inhibition of hsa‐miR‐6817‐5p expression could partially reverse the suppressive effect of si‐circRFWD2 and si‐circINO80 on the osteogenesis. It is suggested that circRFWD2 and circINO80 could regulate hsa‐miR‐6817‐5p and promote recombinant NELL‐1‐induced osteogenesis of hASCs.Although we tried the best to design our experiments, there are some limitations in this study. It was difficult to obtain the clinical samples of unilateral coronal synostosis, where NELL‐1 was detected to be over‐expressed, and thus, we did not verify the expression profiles of CircRFWD2 and circINO80 axis in the clinical specimen data. Furthermore, some interesting and valuable research issues, for example, whether and how NELL‐1 could regulate miR‐6817‐5p, are need to be studied. Therefore, we would focus on miming and enriching the mechanism of circRFWD2 and circINO80 axis in recombinant NELL‐1‐induced osteogenesis, and this study opened the window.5circRFWD2 and circINO80 were identified to up‐regulated during the recombinant NELL‐1‐induced osteogenesis. Mechanistically, circRFWD2 and circINO80 could regulate hsa‐miR‐6817‐5p and influence recombinant NELL‐1‐induced osteogenic differentiation of hASCs. Therefore, circRFWD2 and circINO80 promise to be the potential molecular targets for the regulation of osteogenesis and bone regeneration.The authors declare that there are no conflicts of interest.X. H., J. L. and Z. Z. designed the research. X. H., X. C., B. Z. and G. Z. conducted the research. Y. L. and J. L. contributed to figures and linguistic revision. X. H. and X. C. wrote the paper. All authors reviewed the results and approved the final version of the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


FADS2) gene encodes for the delta-6-desaturase, which is involved in the biosynthesis of C20:4 from linoleic acid (C18:2). The purpose of this study was to characterise mutations in the promoter of the porcine FADS2, evaluating in particular the effect of one haplotype tagging polymorphism (rs321384923A > G) on the biosynthesis pathway of C20:4. A total of 1,192 Duroc barrows with records on fatty acid composition in muscle and subcutaneous fat were genotyped. Pigs carrying the A allele showed, irrespective of fat content, both enhanced FADS2 expression and higher C20:4 in muscle and exhibited increased ratios of C20:4 to C18:2 and of C20:4 to eicosadienoic acid (C20:2) in both muscle and adipose tissue. Despite the inverse relationship observed between C20:4 and fat content, the rs321384923 polymorphism had no impact on lean weight. It is concluded that the haplotype encompassing the rs321384923 polymorphism at the porcine FADS2 affects the n-6 fatty acid profile by specifically modifying the desaturation efficiency of C18:2 to C20:4 rather than by concomitant variations in C18:2 following changes in fat content.Arachidonic acid (C20:4) is related to a wide range of biological effects including lipid homeostasis. The fatty acid desaturase-2 ( C20:4 is essential in many organs such as liver and brain, where it is one of the most abundant fatty acids. In skeletal muscle, C20:4 promotes myocyte growth both in vitro1 and in vivo2 through the Akt/mTOR pathway. Mainly esterified into phospholipids, it also exerts a substantial contribution to maintaining membrane fluidity and in cell signalling. The C20:4 content differs between lipid classes, tissues and muscles and is influenced by both the diet and the individual’s genetic background4. Diets rich in C20:4 or diets producing relatively high levels of linoleic acid  result in enhanced levels of C20:4 in plasma5 and muscle6. Although C20:4 can be taken up from the diet, it can also be synthesised in the animal. There is evidence indicating that the biosynthesis of C20:4 is genetically mediated, notably in pigs, where substantial genetic variation between4 and within genetic types7 has been reported.Arachidonic acid  gene, is responsible for the first and rate-limiting step in the biosynthesis of C20:4 8. The lack of FADS2 leads to obesity resistance and, as reported by Stoffel et al.9 using auxotrophic mice mutants, it may activate a surrogate reaction in which C18:2 is elongated to eicosadienoic acid  and then to eicosatrienoic acid  but not to C20:4. In pigs, FADS2 is located in chromosome 2 (2:9632454–9667044:-1) as a part of a cluster including FADS1 and FADS3. The genomic structure of the gene is comprised of 12 exons and 11 introns (assembly Sscrofa11.1), which produce three protein-coding splice variants. The activity of FADS2 in the synthesis of long chain fatty acid is enhanced by an alternative transcript of pig FADS110, as also reported in baboons and humans11. In a recent genome-wide association study with five divergent pig populations, Zhang et al.12 provide evidence that in Erhualian pigs the region containing FADS2 was associated with the C20:4 content in muscle. Although an uncharacterised polymorphism in exon 3 of the pig FADS2 has been associated with C20:4 and intramuscular fat (IMF) content13, the sequence variation of FADS2 has not been otherwise investigated. FADS2 is a TATA-less gene and such genes are often subjected to complex transcription mechanisms. This makes the promoter region of FADS2 a sensible location for screening for DNA polymorphisms. The aim of our study was to describe genetic variants in the promoter of the porcine FADS2 and then to further investigate their association with C20:4 and fat content in the main lipogenic tissues. To this end, we made use of a biorepository of fat, muscle and liver specimens from a high-fat Duroc pig line where at least two relevant genes for fatty acid composition are also segregating14,15.The delta-6-desaturase enzyme, encoded by 0:4 Fig. , where CAll pigs used in the study were raised and slaughtered in commercial units following applicable regulations and good practice guidelines on the protection of animals kept for farming purposes, during transport and slaughter. The experimental protocol was approved by the Ethical Committee on Animal Experimentation of the University of Lleida.16. In each batch, pigs were raised from 75 days of age until slaughter at 210 days in the same farm under identical conditions. Pigs had ad libitum access to commercial feed . From 160 days of age onwards they were fed a finishing diet including around 6.0% fat . All pigs were slaughtered in the same abattoir, where carcass weight and carcass backfat and loin thickness at 6 cm off the midline between the third and fourth last ribs were measured by an on-line ultrasound automatic scanner . Immediately after slaughter, samples of semimembranosus  muscle, subcutaneous adipose tissue (n = 388) and liver (n = 118) were collected, snap-frozen, and stored at −80 °C. After chilling for about 24 h at 2 °C, samples of the muscles gluteus medius  and longissimus thoracis  were collected, vacuum packaged, and stored at −20 °C. Intramuscular and liver fat content, as well as fatty acid composition, were determined in duplicate by quantitative gas chromatography17. The proportion of C18:2, C20:2 and C20:4 were expressed as percentages relative to total fatty acid content  and their ratios calculated as indicators of FADS2 activity.A total of 1,192 barrows from 159 sires and 590 dams of the same Duroc line were used in this experiment. Pigs were raised in 20 batches between 2002 and 2016 following a similar standard protocol for data recording and tissue samplingFADS2 (~1 Kb) was amplified and sequenced in a subset of 14 pigs with high or low C20:4 content in SM . Thermocycling conditions were 95 °C 10 min, 35 cycles of 95 °C 20 sec, 56 °C 20 sec and 72 °C 20 sec finishing with 72 °C 5 min. Ten μl of PCR were digested with AvaI (37 °C × 3 h) and solved by electrophoresis in agarose gels. Additionally, two other SNPs known to influence fat content and composition in our resource Duroc line (the AY487830:g.2228 T > C SNP at the stearoyl-CoA desaturase (SCD) gene on chromosome 14 and the NM_001024587:g.1987C > T SNP at the leptin receptor (LEPR) gene on chromosome 6) were genotyped as described in Ros-Freixedes et al.15Genomic DNA was isolated from GM muscle samples using a standard protocol. The proximal promoter of the FADS2 and two reference genes, YWHAZ and RPL32, were analysed by a quantitative PCR assay  and 31 livers from one batch  was isolated with TRI-Reagent (Sigma-Aldrich) following the manufacturer’s indications. Purity of the RNA was assessed by spectrophotometry with a Nanodrop-1000 and the integrity was tested by electrophoresis in agarose gels. FADS2 genotypes with C18:2, C20:2, C20:4 and their ratios was performed using a mixed model including the batch (20 levels for fatty acids), the FADS2 genotype (3 levels), the SCD genotype (3 levels) and the LEPR genotype (3 levels) as fixed effects and the sire and the dam as random effects, with fat content as a covariate . The same model was used for gene expression (without the covariate) and for carcass traits (with the age at slaughter as a covariate instead of fat content). Additivity was tested replacing the genotype effect by the covariate  for the AA, AG, and GG genotypes, respectively. The effects of the FADS2 genotype and additivity were tested using the F-statistic while the pairwise differences among FADS2 genotypes were contrasted with the Tukey-HSD test. Results are presented as least-square means ± standard error and were considered statistically significant at P < 0.05. The non-linear relationship of IMF with C20:4 was assessed regressing the reciprocal term of IMF on C20:4. All models were solved using the JMP Pro 12 package .The association analysis of FADS2 gene  binding site while no potential transcription factor binding sites were detected in the other two SNP sites. While human and rat FADS2 expression responds to oestrogen hormone19 and vitamin A20, identification of causal mutations has been hindered by the presence of clusters of polymorphisms in strong linkage disequilibrium both upstream and downstream of the translation start site21.A total of 5 SNP polymorphisms and one 12 bp insertion were segregating in the proximal promoter of the pig FADS2 rs321384923 genotypes by SCD and LEPR genotypes are given in Supplementary Table g.2228 T > C SCD and the g.1987C > T LEPR SNPs were both segregating at intermediate frequencies . All possible genotypes for the three SNPs were observed and, as expected for genes in different chromosomes, they were in linkage equilibrium .The frequencies of the FADS2 expression was determined in SM on pigs of the three FADS2 rs321384923 genotypes . As evidenced by the allele substitution effect , heterozygous pigs displayed intermediate levels of gene expression. Results of FADS2 expression in liver confirmed the same trend, with pigs carrying the A allele (AA and AG) showing higher expression than the GG pigs . From this foundation, we proceed to explore the possible functional consequences caused by the higher expression of the A allele at the porcine FADS2.FADS2 rs321384923 was first assessed in GM , and -0.13 ± 0.03, for C20:2 . The same trend was observed when fatty acids were quantitatively expressed in mg/g of muscle. These values, although only accounting for about 2% of the total variance of these two fatty acids (2.2% for C20:4 and 1.7% for C20:2), provide support to the hypothesis that there exists genetic variation in the promoter region of the FADS2 that impacts n-6 fatty acid biosynthesis in pigs. This was confirmed by analysing the indicator ratios of FADS2 activity .The effect of the FADS2 rs321384923 genotypes with fatty acid composition was investigated in two other muscles (LM and SM), subcutaneous fat and liver. The effect of FADS2 SNP in LM and SM were in line with those observed in GM, particularly for the C20:4 to C18:2 and C20:4 to C20:2 ratios . Likely due to the limited data size, this effect was not evident in SM . No difference between FADS2 genotypes was detected for C18:2 and C20:2 in both LM and SM. The results in LM and SM were the same when individual fatty acids were expressed in mg/g of muscle.The association of ios Fig.  and to Cios Fig. . The C20ios Fig. . HoweverFADS2 rs321384923 with carcass weight and lean percentage and, as a result, with lean weight  when adjusting for IMF  with effect on the desaturation pathways leading to C20:4 biosynthesis. The polymorphism is a tagging SNP for a 3-SNP haplotype in the FADS2 promoter and it is situated -706 bp upstream from the ATG codon and -294 bp upstream from the start of transcription of the closest FADS2 transcript (ENSSSCT00000014289.3). Two previous genome-wide association studies have identified markers around this haplotype region associated with FADS2 expression and fatty acid metabolic traits. In the first one, in an Iberian x Landrace backcross, Revilla et al.22 found that the most significant cis-SNP for FADS2 gene expression (rs81474400) was located 230 Kb upstream of the FADS2. In this study, the three SNPs of the haplotype were found to be segregating in the Iberian founders, but no association was observed between the most proximal of them (rs331050552) and FADS2 mRNA expression in the backcrossed pigs. The low frequency of the minor allele and/or different haplotype structure in the backcrossed individuals could have interfered with the expected results in an experimental population of limited size. In the second genome-wide association study, a SNP (rs81360272) located in the fifth intron of FADS2, was reported to be associated in Erhualian pigs with proxy ratios of FADS2 activity12. SNPs from both studies are from the Illumina’s pig genotyping array and encompass a region containing at least four additional SNPs from this array. Using data on 272 Duroc pigs from our line genotyped with this chip, we found that these two SNPs were in low linkage disequilibrium with our tag SNP (r2 = 0.10–0.15) and had no effect on C20:4 and associated ratios. In contrast, the two nearest upstream SNPs to our haplotype (rs343441264 and rs81360470) were almost fully linked with our tag SNP (r2 = 0.88–0.92) and parallel their effects. Overall, this genomic pattern would confirm that our tag SNP is capturing a functional variant in the promoter region of FADS2 influencing C20:4 content in pigs.We report here a SNP in the promoter region of the pig FADS2 expression and, as a result, the desaturase activity in both muscle and subcutaneous fat. FADS2 is the rate limiting enzyme in the conversion of essential fatty acids C18:2 and α-linolenic to long-chain polyunsaturated fatty acids. The knock out of this gene results in mice lacking polyunsaturated fatty acids beyond eicosatrienoic acid23  and (ii) C20:2 to dihomo-γ-linolenic acid . These are critical steps to produce C20:4. Thus, the presence of the more active A allele accelerates the production of C20:4 through both routes, as seen by significant changes in the C20:4/C18:2 and C20:4/C20:2 ratios, resulting in 12–14% more C20:4 in the three muscles tested  regardless of IMF. Furthermore, this was a consistent additive effect, paralleling the gene expression results. In agreement with this, there was a small correlated decrease in the amount of C18:2 in the genotypes carrying the A allele. These results are in line with previous findings9 indicating that a lack of FADS2 triggers an alternate reaction where C18:2 is diverted to C20:2 instead of all-cis-6,9,12–18:3, the first intermediate fatty acid in the canonical endogenous synthesis of C20:4. In the absence of FADS2, C20:2 cannot be transformed to all-cis-8,11,14–20:3, the last precursor of C20:4, and thus it is further desaturated via FADS1 to all-cis-8,11,14–20:3, an aberrant fatty acid which is incorporated as a surrogate of C20:4 in the diacylglycerol-backbone of membrane phospholipids. Unfortunately, we have no available data to test whether all-cis-8,11,14–20:3 declines as expected with the presence of the A allele.The presence of the A allele of the rs321384923 SNP additively enhances FADS2 than muscle10, the effect of the FADS2 rs321384923 on the accumulation of C20:4 is more evident in muscle than in subcutaneous fat and liver. Apart from having more statistical power in muscle, the functional approach in this tissue can be more accurate for the reason that it shows a relatively higher de novo fatty acid synthesis24. In this regard, the case of the Duroc pigs is particularly interesting because they present a higher proportion of C18:2 in IMF relative to other breeds3. Moreover, an alternative transcript of FADS1, particularly enriched in subcutaneous fat and liver10, has been shown to regulate FADS2 activity in humans11. In this line, in a previous expression genome-wide association study22, the correlation between FADS1 and FADS2 mRNA expression was higher in liver than in subcutaneous fat  while FADS2 expression showed the lowest correlation (r = 0.23) between these two tissues. Thus, the interaction between these two genes and tissue-specific mechanisms of regulation is a question worth exploring in the future.Despite the fact that, in pigs, liver and subcutaneous fat express 8–10 times more et al.25). Diets rich in C18:2 result in an increase in C20:4, which can then be converted into prostacyclins and endocannabinoids, both of them with a strong pro-adipogenic activity25. Indeed, although many of these studies report weight gain and increased adipose inflammation, the final outcome is highly dependent on the whole diet and other external factors such age or physical activity. Some research even showed that dietary supplements with C18:2 increase lean mass26. In our study with pigs none of the carcass traits analyzed were affected by the higher FADS2 activity of the A rs321384923 allele. Even though C20:4 was negatively correlated with carcass weight  and positively with lean content , the effect of the FADS2 SNP on n6-fatty acid composition did not per se modify these traits. Our results also indicate that, although unevenly across muscles, the tag SNP could exert some influence on IMF, especially in muscles displaying lower C20:4 content  than in psoas major (around 10 times), even when both muscles were compared at a similar level of IMF. In a limited subset of pigs, we obtained that in GM C20:4 was around 35 times more abundant in phospholipids than in neutral lipids. These results indicate that the ability of C20:4 to get incorporated into membrane phospholipids, and likely to mediate in cell signaling events, could be muscle-specific. Results in mice evidenced that FADS2 deficiency alters the membrane phospholipidomic profiling, affecting the maturation of transcription factor sterol-regulatory- element-binding protein and therefore lipid homeostasis9. In this sense, an interesting piece of research to address the nuances of functionality of C20:4 in pig muscle is to determine how differently the FADS2 genotypes affect phospholipid and neutral lipid fatty acid composition and whether their allocation relates with fat content. IMF is a relevant trait for the pig industry in general, but particularly in Duroc lines used in premium quality meat markets, where pigs are raised to display a high level of IMF. For this reason, rs321384923 cannot be discarded as a candidate marker to increase IMF without altering lean weight.Many human and mouse studies positively correlates Δ-6 activity with metabolic syndrome, insulin resistance and obesity (reviewed in Naughton 27, our results confirm that the porcine FADS2 is subjected to functional genetic variation while providing evidence that the rs321384923 SNP in its promoter region impacts gene expression. We showed that there is an haplotype tagging SNP in the promoter region of FADS2 that results in a more efficient transformation of C18:2 into C20:4. However, we were not able to observe any consistent implication of this on the traits usually selected for in pig populations. Evidence in humans indicates that fatty acid desaturases affect plasma and tissue lipid profiles and therefore associated disease risk factors. Recent findings in humans suggest that FADS genes have been subjected to strong positive selection in response to C18:2 consumption and that this event is not neutral in relation to plasma cholesterol levels28 and to chronic and inflammatory disorders27. Further studies are needed to identify the molecular mechanisms by which variation in FADS2 modulates gene expression and functional phenotypes. In this regard, the present work confirms that selected pig populations can be an interesting genetic resource.In line with findings in humansSupplementary Information


Cells have developed numerous adaptation mechanisms to external cues by controlling signaling-pathway activity, both qualitatively and quantitatively. The Wnt/β-catenin pathway is a highly conserved signaling pathway involved in many biological processes, including cell proliferation, differentiation, somatic cell reprogramming, development, and cancer. The activity of the Wnt/β-catenin pathway and the temporal dynamics of its effector β-catenin are tightly controlled by complex regulations. The latter encompass feedback loops within the pathway  and crosstalk interactions with other signaling pathways. Here, we provide a review shedding light on the coupling between Wnt/β-catenin activation levels and fluctuations across processes and cellular systems; in particular, we focus on development, in vitro pluripotency maintenance, and cancer. Possible mechanisms originating Wnt/β-catenin dynamic behaviors and consequently driving different cellular responses are also reviewed, and new avenues for future research are suggested.  Wnt proteins are key mediators of cell specification and patterning in development, adult tissue homeostasis, and stemness . Mutatioβ-catenin has a central role in directing diverse intracellular functions. It is involved in cell–cell adhesion through interaction with the E-cadherin cell-adhesion complex and the microtubule network ,32,33,34The amount of β-catenin protein not complexed with E-cadherin is buffered by the destruction complex. The latter is a multiprotein complex; it consists of scaffolding proteins Axin, tumor suppressor APC, the serine–threonine kinases glycogen synthase kinase 3 (GSK3β), and the casein kinase 1 (CK1), and it is responsible for cytosolic β-catenin phosphorylation, ubiquitination, and degradation ,53,54,55http://web.stanford.edu/group/nusselab/cgi-bin/wnt/).Meanwhile, nuclear TCF proteins associate with transcriptional repressors such as Groucho (Gro) and inhibit the expression of β-catenin target genes ,57,58 FA. When aThe ability of the Wnt/β-catenin pathway to co-ordinate cell fate and homeostasis during development and in adult tissue has been extensively studied both in vitro and in vivo (reviewed in Reference ); furtheIn this review, we focus on the specific dose- and dynamic-dependent Wnt/β-catenin signaling roles across systems and organisms.In vivo embryogenesis relies on the interplay of signaling cascades to activate tissue- and organism-specific differentiation programs. Multicellular-organism development relies on gene-expression patterns and on anterior–posterior (AP) polarity dictated by cellular positioning . The WntXenopus, low or high β-catenin accumulation levels in the anterior/posterior endoderm have been shown to maintain a foregut fate or to promote intestinal development, respectively [In ectively ,84. In zectively . lacZ, both the epithelium and mesenchyme of the mandible, maxilla and nasal processes, but the frontonasal area showed Axin2 signal [The importance of controlled β-catenin protein levels is also evident in murine and human hair-follicle homeostasis, where protein absence or overexpression results in inappropriate differentiation and/or tumor initiation ,87. Hair2 signal ,93. More recently, increased β-catenin expression was shown to force the differentiation of the embryonic ectoderm into hair follicles and promote de novo hair-follicle induction in adult skin; on the other hand, β-catenin depletion led to reduced proliferation of epithelial cells and premature catagen (i.e. regression phase prior to telogen) . These oVertebrae formation starts from cellular precursors in a process known as the segmentation clock ,106,107;The mutual regulation of various signaling pathways and the resulting gradients and oscillations of molecules guide cell positioning and control somitogenesis .Notch was the first signaling pathway shown to control the process, as the majority of the oscillatory genes are Notch-dependent ,117,118.Recent in vivo studies from Wilson’s group reported differential levels of Wnt molecules during cell specification. Two subpopulations, both pluripotent, were identified in postimplantation epiblast stem cells (EpiSCs): a partially neuronal-like (Sox1+) fraction, expressing low Wnt/β-catenin levels, and a fraction of progenitor cells, with intermediate activation of the Wnt pathway. Further increase of Wnt/β-catenin signaling activity above a threshold irreversibly promotes mesendodermal and neuromesodermal differentiation . The intestine has a peculiar functional architecture designed to maximize the available surface for absorbing nutrients and water. Epithelial cells invade the surrounding connective tissue to form tubular glands known as “crypts” , which aIntestinal-epithelium regeneration involves a series of events comparable to those taking place during intestinal development at the embryonic stage. ISCs migrate from the bottom of the crypt up to the villi; during this upward migratory process, cells are subjected to different stimuli blocking cell proliferation and promoting differentiation into all cell types required for intestinal functions  ,133. In The Wnt/β-catenin pathway has been demonstrated to control, in a dose-dependent manner, intestinal epithelium homeostasis in both health and disease. Dickkopf-1 (Dkk-1)-mediated Wnt pathway inhibition and Wnt pathway ablation are detrimental for crypt fission both in vitro and in vivo ,136, andIn the crypt, Wnt acts in coordination with other signaling pathways , namely:Wnt molecules and gradients regulate different aspects of nervous system development and function in vertebrates.Exogenous overexpression of a stable  form of β-catenin in hippocampal neuronal cultures causes an increased number of dendritic branches in a dose-dependent manner . To testThe canonical Wnt pathway also guides synaptogenesis ,156,157:The use of a β-catenin-activated promoter driving the expression of the β-galactosidase reporter helped in defying neurons that respond to activated β-catenin during mouse development. This study confirmed activation of the Wnt pathway in the mid-hindbrain and in the limb apical ectodermal ridge, and identified additional activated regions like the notochord and brain endothelia . Embryonic stem cells (ESCs) are characterized by pluripotency  and self-renewal . Pluripotency is a transient state in vivo; instead, ESCs can indefinitely be expanded in vitro, maintaining either ground state/naive or primed pluripotency states if isolated from the pre- or postimplantation epiblast, respectively . CultureIf and how β-catenin transcriptional activity is relevant for pluripotency is debated: in basal conditions  it is negligible , and β-cThe canonical Wnt pathway is also relevant for somatic cell reprogramming . Wnt coRegarding human embryonic and induced pluripotent stem cells , recent studies indicate that their pluripotent state resembles that of mouse EpiSCs , as theyLacZ Wnt reporter mouse model showed differential sensitivity in reporter activity when stimulated with the canonical Wnt pathway. Furthermore, when studying hematopoietic stem cell (HSC) differentiation in the presence of varying Wnt/β-catenin activity, mild/intermediate levels were shown to enhance clonogenicity and myeloid differentiation, while high levels strongly reduced the number of colonies; only a mild increase was able to confer increased HSC repopulation potential [WNTs and their effectors are also crucial for potency maintenance in adult stem cells ,196,197.otential . Homeostasis tightly controls the number of cells within tissue, balancing cell growth and survival. Genetic mutations or sporadic events can result in uncontrolled cell proliferation and/or increased cell survival, which can ultimately lead to cancer initiation ,200. OftColorectal cancer (CRC) is the first and most characterized cancer model involving the canonical Wnt pathway. Because of APC mutations, β-catenin levels increase, and pro-proliferative genes are activated following its nuclear translocation ,202. A sA study on a cohort of human gastric adenocarcinomas revealed β-catenin nuclear accumulation in 29% of samples, whereas 71% only showed membrane β-catenin staining; this study did not investigate a possible correlation between β-catenin levels and/or intracellular localization and cancer severity . SimilarThe role of β-catenin in cancer is not only restricted to the proliferative advantage of cancer cells, but also to their ability to colonize surrounding tissue, potentiating metastasis formation ,219,220 Although β-catenin is involved in many phases of cancer progression and higher levels worsen prognosis ,226, litThe pleiotropic roles of the Wnt/β-catenin pathway in regulating multiple processes, including embryogenesis, pluripotency, differentiation, and cancer, have been reviewed. It appears clear that the functions of the pathway are highly cell- and context-dependent ; across Feedback loops in pathway topology ,228 and Xenopus embryos data [Mathematical models can be instrumental in providing quantitative insights into the characteristics and dynamics of signaling pathways and coupled dynamic processes, allowing to test hypotheses and generate in silico predictions . While tyos data ) that thyos data . The canyos data ,240 for yos data  have shoyos data ,242,243.Novel experimental techniques can also be instrumental to developing a better and quantitative understanding of the role of spatial and temporal Wnt/β-catenin  pathway dynamics in controlling tissue homeostasis. Three-dimensional in vitro cell clusters and organoids enable studying processes like organogenesis and cancer development in a fully controllable setting , especiaLive-cell imaging, combined with precise perturbations of signaling-pathway dynamics, might contribute significantly to dissect how the latter control cell fate. In a recent work, a microfluidics/imaging-based approach was developed to entrain oscillations of Notch and Wnt signaling to predetermined external periodic forces in the anterior monolayer PSM ; this exThe use of feedback control, recently combined with microfluidics/imaging platforms to precisely regulate gene expression in living cells , and wit


Pentalonia nigronervosa) in a persistent, non-propagative, circulative manner. Interaction of BBTV and banana aphid had been studied in several ways, such as transmission and translocation of BBTV inside the banana aphid body at cellular level. However, the molecular mechanism underlying the interaction between BBTV and banana aphid have been poorly understood. Therefore, this transcriptomic study was conducted to obtain the raw data for differential genes expression study in BBTV viruliferous (Vr) and non-viruliferous (NVr) banana aphid. Here, we present two data sets of RNA seq raw reads which is available in GenBank Sequence Read Archive (SRA) database with accession number of SRX6918251 and SRX6918252 for the Vr and NVr banana aphid respectively.Banana bunchy top disease (BBT) is one of the most economically serious viral diseases of banana caused by banana bunchy top virus (BBTV: Nanoviridae: Babuvirus). BBTV is a circular, ssDNA virus which is suitable in the phloem tissue and currently only being transmitted by the banana aphid ( Methods of insect rearing and collection, total RNA extraction, sequencing and generating clean transcriptome data is presented in the following section.22.1Initial colony of banana aphids were obtained from banana plantation grown in Bantul, Yogyakarta, Indonesia, and further transferred to the greenhouse facilities in Universitas Gadjah Mada, Yogyakarta. BBTV infected and non-infected banana were utilized for the rearing of BBTV Vr and NVr banana aphid respectively. New banana seedlings were continuously provided to replace old banana seedling to keep banana aphid population exist. Pool of 40 individuals banana aphid at different instars were collected and immersed on RNA later (Ambion) for RNA extraction. Confirmation of the BBTV viruliferous banana aphid population was conducted by amplifying the BBTV primers BBT1: 5′-CTCGTCATGTGCAAGGTTATGTCG-3′ and BBT 2: 5′-GAAGTTCTCCAGCTATTCATCGCC-3′, on pool of 10 adult aphid DNA (Geneaid DNA extraction kit) targeting 250–350 bp PCR product [2.2Both Vr and NVr banana aphid samples (whole body) were RNA extracted using RNeasy Plus kit  according to the manufacturer's instructions. The quantity and quality of the total RNA were validated using NanoDrop spectrophotometer  for the purity of the RNA samples, and Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit) for the RNA integrity , 28S/18S and the fragment length distribution. The samples were further sequenced using BGISEQ-500 platform following the steps as follow; 1) mRNA enrichment, 2) RNA fragment and reverse transcription: 3) End repair, 4) PCR amplification, 5) Denature and cyclization, 6) Sequencing on BGISEQ- 500 platform.2.3Filtering step was first performed on the raw sequencing reads generated by RNA-seq., mainly by removing those raw reads with adaptors and reads with more than 5% of unknown bases (N). After filtering, the remaining reads are called “Clean Reads” and stored in FASTQ file. Those clean read then ready for further assembly process. Descriptive statistics on the RNA-seq data of the two set of both Vr and NVr banana aphid samples are given in


H. parasuis. In PK-15 and NPTr cells, it was also confirmed that infection with a high-virulence strain of H. parasuis induced cytoplasmic accumulation and nuclear translocation of β-catenin. H. parasuis infection caused a sharp degradation of E-cadherin and an increase of the epithelial cell monolayer permeability, as well as a broken interaction between β-catenin and E-cadherin dependent on Wnt/β-catenin signaling pathway. Moreover, Wnt/β-catenin signaling pathway also contributed to the initiation of epithelial-mesenchymal transition (EMT) during high-virulence strain of H. parasuis infection with expression changes of epithelial/mesenchymal markers, increased migratory capabilities as well as the morphologically spindle-like switch in PK-15 and NPTr cells. Therefore, we originally speculated that H. parasuis infection activates the canonical Wnt/β-catenin signaling pathway leading to a disruption of the epithelial barrier, altering cell structure and increasing cell migration, which results in severe acute systemic infection characterized by fibrinous polyserositis during H. parasuis infection.In this study, animal experimentation verified that the canonical Wnt/β-catenin signaling pathway was activated under a reduced activity of p-β-catenin (Ser33/37/Thr41) and an increased accumulation of β-catenin in the lungs and kidneys of pigs infected with a highly virulent strain of  Haemophilus parasuis is the etiological agent of Glässer's disease, an important bacterial disease in swine worldwide, which causes serious economic loss to the global pig industry  and Newborn pig tracheal (NPTr) cells. Previous studies demonstrate that PK-15 cells are suitable models for studying H. parasuis-induced inflammation and immunity , cyclooxygenase2 (COX2), and plasminogen activator inhibitor-1 (PAI-1), which are involved in cell apoptosis, proliferation, carcinogenic effects, cell migration, cell adhesion .Nine 30-day-old piglets were randomly divided into three treatment groups equally. Three groups of piglets were injected intraperitoneally with 1 ml normal saline as control, 2 × 102O2 (Sigma-Aldrich). After repairing antigen with microwave for 30 min, the sections were blocked with 5% bovine serum albumin (Sigma-Aldrich) for 20 min and incubated with primary antibody anti-β-catenin (at a dilution of 1:100)  at room temperature for 2 h. After washing 3 times with PBS (pH7.4), the sections were incubated with secondary antibody for 30 min and then treated with Strept Avidin-Biotin Complex  and 3,3′-diaminobenzidine  after washing with water. The nucleus was counterstained with hematoxylin for 30 s. The results were carried out with Nikon 80i microscope .Samples from lungs and kidneys were embedded with paraffin after fixed in paraformaldehyde overnight, and then tissues were cut at the thickness of 4 μm to perform immunohistochemistry. The sections were deparaffinized, rehydrated and treated with 3% H2 without any antibiotics. H. parasuis SH0165 strain  and HN0001 strain  were cultured in tryptic soy broth  supplemented with 10% newborn calf serum (NBCS) (Gibco) and 0.2 mg/ml nicotinamide adenine dinucleotide (NAD)  without any antibiotics. After incubation at 37°C with circular agitation (200 rpm) overnight, bacteria were harvested by centrifugation at 5,000 × g for 5 min, washed with PBS for three times and resuspended in fresh DMEM medium to infect cells in vitro. Moreover, we have tested the numbers of bacteria at 6 h, 12 h, 24 h after adding into cells, there was no significant difference in quantity of bacteria whether the inhibitors were added into medium  and new-born pig trachea (NPTr) cells were separated from piglets refer to the method of Ferrari . Monoclonal antibodies against E-cadherin (sc-8426) and polyclonal antibodies against β-catenin (sc-7199) were purchased from Santa Cruz Biotechnology (USA). Polyclonal antibodies against phospho-β-catenin (Ser33/37/Thr41) (#9561) and monoclonal antibodies against Caveolin-1 (#3267), PCNA (#2586) and Vimentin (#5741) were purchased from Cell Signaling Technology (USA). Monoclonal antibodies β-actin (#AA128) and GAPDH (AF1186) were obtained from Beyotime (China). Lithium chloride (LiCl) (L7026) was obtained from Sigma-Aldrich (USA). TGF-β1 (100-21-2) was obtained from Peprotech (USA). Alexa Fluor 488-labeled Goat Anti-Rabbit IgG(H+L) (A0423) and Alexa Fluor 555-labeled Donkey Anti-Mouse IgG(H+L) (A0460) were purchased from Beyotime (China). Wnt/β-catenin inhibitor ICG001 (S2662), IWR-1-endo (S7086) were obtained from Selleck (USA). IWR-1-endo inhibits Wnt signaling by inhibiting Wnt-induced accumulation of β-catenin and ICG001 antagonizes Wnt/β-catenin/TCF-mediated transcription and specifically binds to CREB-binding protein (CBP)  following the manufacturer's instructions.To assess the activation of Wnt/β-catenin pathway, cells seeded in 96 well plates were transfected with 0.1 μg/well of either TOPflash or FOPflash and 0.1 μg/well of pRL-TK by 0.5 μg/well Lipofectamine 2000  following the manufacturer's instructions. At 24 h after transfection, cells were treated or untreated with various inhibitors for 2 h before stimulation with Total protein of 0.1 g tissue samples of each tissue was extracted by 1 mL RIPA Lysis Buffer  supplemented with 1 mM protease inhibitor cocktail (Roche). Protein concentration was calculated by BCA assays  following the manufacturer's instructions and 15 μg protein of each sample was used for western blot analysis.H. parasuis or LiCl. Cells were harvested at the indicated time points by 200 μL/well Cell lysis buffer for Western and IP  supplemented with 1 mM protease inhibitor cocktail (Roche). The subcellular fractionation of proteomic samples from membrane, cytoplasm, and nucleus was obtained by Qproteome cell compartment kit (Qiagen) following the manufacturer's instructions. Western blot analysis was performed as described previously  for 3 times and fixed with 4% paraformaldehyde (Sigma) for 15 min. The cells were then permeabilized with 0.1% Triton X-100 (Sigma) for 15 min followed by blocking with 5% bovine serum albumin (Sigma-Aldrich) for 1 h at room temperature. After blocking, cells were incubated with primary antibody for 2 h and then incubated with AlexaFluor-conjugated secondary antibodies for 1 h after washed by PBS for 3 times. Prior to imaging, nucleus is counterstained with 4′-6-diamidino-2-phenylindole (DAPI) (Invitrogen). Coverslips were imaged at 40× by a Zeiss LSM 510 Meta confocal microscope .For all indirect immunofluorescence analysis, cells were seeded on 13-mm sterilized coverslips and cultured overnight to 50–60% confluence. Cells were treated or untreated with indicated inhibitors for 2 h, and then stimulated with 2 of surface area and 0.4 μm of pore size, Costar, USA) and cultured for 48 h to form confluent monolayers with a resistance of 300 Ω*cm2. Cells were subsequently treated or untreated with indicated inhibitors (10 μM) for 2 h, and then stimulated or unstimulated with H. parasuis (108 CFU/mL) or LiCl (20 mM). At 0, 6, 12, 18, and 24 h post-infection, the EVOM2 volt ohm meter  was used to measure the transepithelial electrical resistance (TEER) of cell monolayers according to the manufacturer's recommendations. Three independent experiments were performed.PK-15 or NPTr cells were seeded on upper compartment of transwell cell culture system  at the indicated periods of time post-infection, 1 μg RNA of each sample was reverse transcribed into cDNA with iScript cDNA synthesis kit (Bio-Rad) and the quantitative real time PCR reactions were performed by SYBR Green Supermix (Bio-Rad) following the manufacturer's instructions. Transcripts of three individual samples were normalized to the corresponding porcine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level. The sequences of the primers are shown in Table SH0165 (108 CFU/mL) or TGF-β1 (10 ng/mL) for 12 h. Wnt/β-catenin inhibitor ICG001 was added 2 h before infection. For the wound healing assay, cells were seeded in 6 well plates and cultured to 90% confluence, and then the monolayer of cells was wounded with sterile pipette tips. The images were observed with an inverted microscope  after treatment described above. Each experiment was performed at least three times independently.PK-15 or NPTr cells were cultured in 60-mm plates to 90% confluence. The morphological changes of cells were photographed with an inverted microscope  after stimulated by Sequence-specific small interfering RNAs (siRNAs) of three genes and siNegative oligonucleotide (negative control) were purchased from GenePharma (China). Cells seeded at 24 well plates were cultured to 70% confluence and transfected with 15 pmol/well sequence-specific siRNAs or siNegative by 1.5 μg/well lipofectamine 2000 (Invitrogen). Twenty-Four hours after transfection, cells were collected to perform qRT-PCR as described above. The primers used in qRT-PCR were presented in Table p-value of < 0.05 was considered significant and a p-value of < 0.01 was considered highly significant.Data are presented as mean ± SD. Data among groups were compared with one-way analysis of variance (ANOVA). A in vivo after H. parasuis infection, we examined the expression of β-catenin in kidney and lung tissues of pigs following infection with the high-virulence H. parasuis strain SH0165 and the non-virulent strain H. parasuis HN0001. As shown in Figures H. parasuis infection with the high-virulence strain, but not the non-virulent strain, activated the Wnt/β-catenin signaling pathway in vivo.To investigate the role of the Wnt/β-catenin signaling pathway H. parasuis strain could activate the Wnt/β-catenin signaling pathway in vivo as described above. Next, we evaluated the effects of Wnt/β-catenin signaling in both PK-15 cells and NPTr cells during H. parasuis infection. LiCl is an activator of Wnt signaling that inhibits the activity of GSK3β, thus stabilizing β-catenin and activating a downstream signal for nuclear translocation , glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and caveolin-1 were, respectively, used as markers for nuclear  or LiCl (20 mM). As shown in Figures H. parasuis infection.Considering that β-catenin/E-cadherin based anchoring junctions are important for AJs, which in turn maintain epithelial integrity   or TGF-β1 (10 ng/mL). The morphological change was further demonstrated by confocal  or TGF-β1 (10 ng/mL). This result is accordance with previous reports depicting the rearrangement of vimentin during EMT process  or TGF-β1 (10 ng/mL) substantially increased the cellular migration compared with the untreated group   and an increased accumulation of β-catenin in lung and kidney tissues of pigs infected with a high-virulence strain of y Figure . IncreasH. parasuis infection brought epithelial barrier disruption with a reduction in E-cadherin. However, we could not determine the exact role of Wnt/β-catenin signaling in regulating the expression of E-cadherin. E-cadherin could be down-regulated by inhibiting E-cadherin expression or cleavage/degradation. The IWR-1-endo is an upstream inhibitor of the Wnt pathway, and its inhibitory effect may be associated with (i) the so-called destruction complex consisting of GSK3β, Axin, and APC, (ii) accumulation of β-catenin in space, (iii) nuclear β-catenin/LEF mediated gene transcription, while ICG001 only targets the latter. Not surprisingly, treatment with inhibitor ICG001 or IWR-1-endo remarkably depressed the reduction of E-cadherin induced by H. parasuis infection almost to the same level, suggesting that this was due to nuclear β-catenin/LEF mediated suppression of E-cadherin expression (Figure H. parasuis infection may result in the transition of epithelial cells to EMT phenotype depending on Wnt signaling pathway.Our results showed that Wnt/β-catenin pathway activated by n Figure . This oun Figure . It was n Figure , suggestH. parasuis induced the transcription of Wnt target genes including MMP7, COX2, PAI-1 in cells, which were reported associated with cell structural change of the cells and the process of EMT. For example, the cleavage of E-cadherin by MMP-7 mediated epithelial cell proliferation and loss of cell-cell contact (Lynch et al., H. parasuis; COX2 has been associated with HIV Tat-induced up-regulation of tight junction protein and disruption of the integrity of the blood-brain barrier (Pu et al., H. parasuis infection (Figure H. parasuis infection depended on Wnt signaling pathway in epithelial cell.Furthermore, we demonstrated that H. parasuis infection. It was found that the mRNA expression of typical EMT transcription factor Snail was markedly up-regulated during H. parasuis infection depending on Wnt signaling pathway (Figure Evidences including the expression changes of selected EMT markers, morphological changes and increased migratory capabilities from this study Figure  confirmey Figure , E-cadhey Figure , showed y Figure , and cany Figure . All thoy Figure . In all,y Figure . This kiy Figure . Previouy Figure , but sely Figure .H. parasuis infection activated the canonical Wnt/β-catenin signaling pathway leading to the disruption of adherens junctions with a sharp degradation of E-cadherin as well as an increase in the permeability of epithelial cells, and initialized EMT process dependent on Wnt/β-catenin signaling pathway in epithelial cells, which involved the expression changes of selected EMT markers, morphological changes and increased migratory capabilities. All these phenotype changes depending on Wnt signaling pathway contributed to disrupting epithelial barriers, altering cell structure and increasing cell migration, which may result in severe acute systemic infection characterized by fibrinous polyserositis during H. parasuis infection.In conclusion, HJ and KH designed the research. RL, HJ, HZ, RZ, and DB conducted the experiments. KH, YL, RH, and YC acquired the data. HJ, RL, KH, and YL analyzed the data. XH and RZ provided the essential materials. HJ, HZ, and KH wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, and 3-[2-(2-aminoethylamino)ethylamino]-propyl-trimethoxysilane with different chain lengths. Firstly, a set of pure epoxy resin models, and four sets of SiO2/EP composite models were established. Then, a reasonable structure was obtained through a series of optimizations using molecular dynamics calculations. Next, the mechanical properties, hydrogen bond statistics, glass transition temperature, free volume fraction, and chain spacing of the five models were studied comparatively. The results show that doped nano silica particles of surfaces grafted with 3-aminopropyltriethoxysilane, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, and 3-[2-(2-aminoethylamino)ethylamino]-propyl-trimethoxysilane with different chain lengths enhanced mechanical properties such as elastic modulus, shear modulus, and volume modulus obviously. The glass transition temperature increased by 15–16 K, 40–41 K, and 24–27 K, respectively. Finally, the data show that the cross-linked epoxy resin modified by nanoparticles grafted with N-(2-aminoethyl)-3-aminopropyl trimethoxysilane had better effects for improving thermo-mechanical properties by the comparatively studying the five groups of parameter models under the same conditions.In this paper, a molecular dynamics simulation method was used to study the thermo-mechanical properties of cross-linked epoxy resins doped with nano silica particles that were grafted with 3-aminopropyltriethoxysilane,  Epoxy resin (EP) has good mechanical properties, thermal stability, and electrical properties ,2. It is2/EP composite models were established. Then, the thermo-mechanical properties of cross-linked epoxy resins were predicted by microscopic indexes such as elastic modulus, volume modulus, shear modulus, hydrogen bonds, glass transition temperature, free volume fraction, and chain spacing. Finally, the aminosilane coupling agent with the best improving properties was selected.In this work, based on molecular dynamics, the effects of three aminosilane coupling agents, with different lengths, on the thermo-mechanical properties of cross-linked epoxy resin were studied. Firstly, a group of pure epoxy resin and four groups of SiO2 composite model (the surface of SiO2 without any modifier), and surface of the silica respectively grafted with epoxy resin doped with 3-aminopropyltriethoxysilane (A1100), N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (A1120), and 3-[2-(2-aminoethylamino)ethylamino (A1130) were built, which were represented as pure, SiO2, A1100, A1120, and A1130. In this paper, COMPASS [2 composites and the pure epoxy resin model. The specific steps to establish the models were as follows:In this paper, molecular dynamics simulations were performed using Materials Studio software. The pure epoxy resin model, the EP/SiOysilane A00, N- Bisphenol A epoxy resin (DGEBA) and 1,3 benzenediamine (BD) hardener monomer were constructed by Forcite module, and a box with the 50 × 50 × 50 Å.6 g/cm3 , and the.6 g/cm3 ,20.(2) The mechanical properties of cross-linked epoxy resins were affected by the size of nanoparticles ,22, so s3, then the cross-linked modeling could be obtained by repeating step (1). Finally, the NVT and NPT were conducted at 300 K to obtain a global equilibrium structure.(3) The aminosilane coupling agents with different chain lengths were grafted on the nanoparticles before being constructed in step (2). In general, the stress-strain behavior in linear-elastic materials  of solidV stands for volume, km and ku are the mass and velocity of the kth particle, kth and the lst particle, kth and the 1st particle. The lame coefficients  follows :(3)λ=16(E), volume modulus (K), shear modulus (G) can be obtained using the following equations:The elastic modulus (E), shear modulus (G), and volume modulus (K) of the five statistical models at different temperatures. The mechanical properties of several models fluctuated with the rise in temperature, which was consistent with previous reports [2 models. The results show that it is necessary to graft silane coupling agents on the surface of nanoparticles to improve the mechanical properties of epoxy resin. In the three groups of nanoparticles grafted with aminosilane coupling agents, the mechanical properties of the A1120 model were significantly higher than those of the A1100 and A1130. Moreover, with the increase in temperature, the mechanical parameters of the A1120 model declined slightly, indicating that the mechanical properties of the A1120 modified nano silica doped epoxy resin were better than the A1100 and A1130.In this paper, the mechanical performance parameters of five models at 300–650 K were calculated.  reports . MeanwhiHydrogen bonding generally refers to a bond formed between a highly electronegative atom in a molecule and another electronegative atom, which produces a certain binding force through the H atom. In classical mechanics definition of hydrogen bond ,26, it h2. At the same time, the number of pure hydrogen bonds is obviously the smallest. Among the three models of grafting the aminosilane coupling agents to the surface of nanoparticles, the A1120 had the most hydrogen bonds at the same temperature, A1100 was the second, and A1130 was the smallest. The results show that the toughness of the epoxy resin was enhanced by doping nanoparticles. Furthermore, the mechanical properties of the cross-linked epoxy resin were improved by grafting A1130 onto the surface of doped nanoparticles, but the toughness was sacrificed compared with the grafted A1100.gT) is a significant parameter that reflects the thermal properties of polymer materials. When the temperature is below gT, the material is in a glassy state, and when the temperature is above g,T the material is in a high elastic state. In general, the transition state between the glass state and the high elastic state is called the glass transition, and the corresponding temperature at the time of the transition is called the glass transition temperature. The glass transition temperature can be obtained by linear fitting of density and temperature [gT can also be obtained by fitting curves of free volume and temperature [2, A1100, A1120, and A1130 were 409 K, 418 K, 424 K, 450 K, and 436 K, respectively. From the statistical results, the glass transition temperatures of the grafted silane coupling agent models were significantly better than those of the pure epoxy resin and SiO2 models. Among them, the A1120 had the highest glass transition temperature, where the gT of A1100, A1120, and A1130 increased by 15 K, 41 K, and 27 K respectively compared with pure epoxy resin. The results indicate that the aminosilane coupling agents could improve the thermal properties of the cross-linked epoxy resin. In some examples within the literature [gT from 400 K to 483 K. Different epoxy resin monomers, curing agents, and experimental conditions make the gT different to some extent, which indicates that the simulation in this paper has certain reliability.The glass transition temperature  and occupied volume (oV). The free volume fraction can be calculated as follows:The free volume not only can reflect the stacking capacity of cross-linked epoxy resin molecules, but also predicts thermal and mechanical properties . In this2, and the free volume fraction of cross-linked epoxy without any nanoparticles was the most. The value of the free volume fraction increased with the increase in temperature, which reflects an increase in the molecular chain motion space. However, the large free volume fraction would result in a decrease in the thermo-mechanical properties of epoxy resin [xy resin . At the 2, A1100, A1120, and A1130 obtained by fitting the free volume-temperature curves were 409 K, 420 K, 425 K, 449 K, and 433 K, respectively. gT of A1100, A1120, and A1130 obtained by volume-temperature fitting curve enhanced by 16 K, 40 K, and 24 K, respectively. This result was close to the glass transition temperature obtained from the previously calculated density-temperature fitting curve. Therefore, the reliability of the calculations in this paper is demonstrated again.In this paper, in order to study the reason why the doped nanoparticles grafted with aminosilane coupling agents of different lengths caused different thermos-mechanical properties of the cross-linked epoxy resin, X-ray was used to study the chain spacing of A1100, A1120, and A1130 models at the temperature of 300 K. The chain spacing of the composite reflects the stacking state of the polymer chain . When X-θ and cutoff was 5~45 Å and 50 Å, respectively. Where θ is the diffraction angle corresponding to the peak of the diffraction curve. θ of A1100, A1120, and A1130 were 16.3 degrees, 18.5 degrees and 18.2 degrees.Using the scattering function in Forcite and X-ray as radioactive source, XRD spectra is calculated. The diffraction parameters were set as follows: The range of 2The increase of chain spacing of polymers would make the density decrease and the hydrogen bond break. The results of the chain spacing calculated from In this paper, the thermo-mechanical properties of cross-linked epoxy resin doped with nano silica particles were studied using molecular dynamics methods. The effects of nano silica particles surface grafted with three aminosilane coupling agents with different chain lengths, which were doped into cross-linked epoxy resin were comparatively studied. The obtained conclusions are as follows:gT when compared to the pure epoxy resin model and epoxy resin model directly doped by untreated SiO2. Finally, an obvious result was obtained showing that among the three aminosilane coupling agents with different chain lengths, the cross-linked epoxy resin modified by nanoparticles grafted with N-(2-aminoethyl)-3-aminopropyl trimethoxysilane had better effects for improving thermo-mechanical properties, which may provide a reference for subsequent modifications of aminosilane coupling agents.Doping nano silica particles can improve the mechanical properties and thermal stability of cross-linked epoxy resin. Moreover, the simulation results showed that nanoparticles surface grafted with aminosilane coupling agents into cross-linked epoxy resin can effectively improve the mechanical properties and thermal stability of cross-linked epoxy resin. Nanoparticles grafted with three amino-silane coupling agents with different chain lengths did not only effectively improve the mechanical properties such as elastic modulus, shear modulus, and volume modulus, but also improved thermal stability such as enhancing the toughness effectively, and increasing the