WNT/β-catenin signaling is upregulated in inflammatory processes and oxidative stress and in many cancers, although there are some exceptions for cancers. The opposite is observed with PPARγ, which is generally downregulated during inflammation and oxidative stress and in many cancers. This helps to explain in part the opposite and unidirectional profile of the canonical WNT/β-catenin signaling and PPARγ in these three frequent and morbid processes that potentiate each other and create a vicious circle. Many intracellular pathways commonly involved downstream will help maintain and amplify inflammation, oxidative stress, and cancer. Thus, many WNT/β-catenin target genes such as c-Myc, cyclin D1, and HIF-1α are involved in the development of cancers. Nuclear factor-kappaB (NFκB) can activate many inflammatory factors such as TNF-α, TGF-β, interleukin-6 (IL-6), IL-8, MMP, vascular endothelial growth factor, COX2, Bcl2, and inducible nitric oxide synthase. These factors are often associated with cancerous processes and may even promote them. Reactive oxygen species (ROS), generated by cellular alterations, stimulate the production of inflammatory factors such as NFκB, signal transducer and activator transcription, activator protein-1, and HIF-α. NFκB inhibits glycogen synthase kinase-3β (GSK-3β) and therefore activates the canonical WNT pathway. ROS activates the phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling in many cancers. PI3K/Akt also inhibits GSK-3β. Many gene mutations of the canonical WNT/β-catenin pathway giving rise to cancers have been reported . Conversely, a significant reduction in the expression of PPARγ has been observed in many cancers. Moreover, PPARγ agonists promote cell cycle arrest, cell differentiation, and apoptosis and reduce inflammation, angiogenesis, oxidative stress, cell proliferation, invasion, and cell migration. All these complex and opposing interactions between the canonical WNT/β-catenin pathway and PPARγ appear to be fairly common in inflammation, oxidative stress, and cancers.Inflammation and oxidative stress are common and co-substantial pathological processes accompanying, promoting, and even initiating numerous cancers. The canonical WNT/β-catenin pathway and peroxisome proliferator-activated receptor gamma (PPARγ) generally work in opposition. If one of them is upregulated, the other one is downregulated and  Cancer is a complex process that can be defined in term of three steps: initiation, promotion, and progression . SeveralOxidative stress also operates at these stages by promoting DNA damages and genes mutations . In receThe canonical WNT/β-catenin pathway regulates several signaling pathways involved in development and tissue homeostasis. This pathway is modulated from transcription level regulations to post-transcriptional modifications. An aberrant WNT/β-catenin pathway is observed in cancers , 14. ThiIn contrast, peroxisome proliferator-activated receptor gamma (PPARγ) is downregulated in numerous cancers . By reguIn numerous tissues, canonical WNT/β-catenin pathway activation induces inactivation of PPARγ, while PPARγ activation induces inhibition of canonical WNT/β-catenin signaling . In mostPeroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors. Four subtypes of PPARγ have been identified: PPARγ1, PPARγ2, PPARγ3, and PPARγ4 , 22. AftThe WNT pathway is involved in numerous pathways that control tissue homeostasis and embryogenesis development. WNT ligands belong to the family of genes observed in humans, Xenopus, mice, drosophilia, and Zebrafish . DysreguCanonical WNT signaling is characterized by the interaction between WNT ligand and specific targets resulting in cytosolic β-catenin accumulation and then its nuclear translocation Figure . The nucThe “on state” is characterized by modified WNT/β-catenin signaling. WNT ligands bind Frizzled  and low-density lipoprotein receptor-related protein 5 (LRP 5/6) co-receptors . Then, DIn several diseases, the WNT/β-catenin pathway and PPARγ act in an opposite manner as in cancers, such as gliomas , 50, neuThe WNT/β-catenin pathway and PPARγ interact through a TCF/LEF β-catenin domain and a catenin-binding domain within PPARγ –64. DownIndeed, in numerous diseases, β-catenin signaling decreases PPARγ expression –80. In mPeroxisome proliferator-activated receptor gamma agonists are considered as a promising treatment through the action of this crosstalk . TroglitNumerous inflammatory cytokines, chemokines, or intracellular pathways, such as the canonical WNT/β-catenin signaling, TNF-α, interleukin (IL)-1, and IL-13, downregulate PPARγ expression –96. The Even if, the molecular mechanisms by which TZDs regulate differentiation and stemness programs have been well studied in adipocytes and normal cells, in cancer cells, they still remain unclear . In normCancer progression is promoted by an environment rich in inflammatory factors, DNA damages, and genetic or epigenetic mutations .Several studies have shown that prolonged inflammation leads to DNA damages and tissue injury . ChronicThe link between inflammation and cancer initiation has been examined in a recent study . InflammPathogenic stimuli can stimulate inflammation and then eradicate the normal host defense response . PathogeThe inflammatory response is regulated by the canonical WNT/β-catenin pathway . MoreoveSeveral inflammatory factors can facilitate the migration and invasion of neoplastic cells . Tumor nIn parallel, chronic inflammation stimulates the expression of cyclooxygenase 2  . NumerouThe STAT3 pathway involved in metastasis, proliferation, and angiogenesis  is activ+. Then, the transfer of electrons through the mitochondrial membrane reduces molecular oxygen and produces the superoxide anion as a primary product. ROS production has a major role in several pathways and in changes of intracellular and extracellular environmental conditions  , 136. ROnditions .Reactive oxygen species are produced by dysregulation of the mitochondrial respiratory chain . During Leukocytes during inflammation are recruited from the damage sites and this leads to an increased uptake of oxygen, which induces the release of ROS and subsequently its accumulation , 149.Several redox-regulated transcription factors have a key role in the stimulation of pro-inflammatory mediators, such as NFκB, a signal transducer and activator of transcription (STAT), activator protein-1 (AP-1), and the hypoxia-inducible factors (HIF) . The oxiNADPH-oxidase (NOX) is stimulated by inflammation and leads to oxidative stress and alteration of nuclear signaling . ROS, acA positive crosstalk between WNT/β-catenin and NFκB has been reported recently . The oveNuclear factor-kappaB inhibits GSK-3β and positively regulates the β-catenin signaling , 169. InNuclear factor-kappaB signaling can regulate WNT/β-catenin signaling through the use of IKKα  and RelAReactive oxygen species production activates the PI3K/Akt pathway, which is overactivated in numerous cancers . PTEN isA recent study by Zhang et al.  has showSeveral genetic mutations lead to the aberrant activation of the canonical WNT/β-catenin pathway . In numeMutations of CTNNB1, a β-catenin target, have been shown to be involved in the initiation of colon, gastric, ovarian, pancreatic, and prostate cancers, but also in melanoma and medulloblastoma , 181. APSeveral studies have shown an interaction between miRNAs and the canonical WNT/β-catenin pathway, such as in osteoblast differentiation and cardiac and bone formation –186 Fig. An aberCancer-stem cells theory is characterized by the fact that cancer cells are derived from certain populations of cells, which possess stem cell properties –191. SevThe role of miRNAs in the regulation of CSCs is currently being investigated. Nevertheless, it has been observed that several miRNAs, such as miR410, can promote tumor growth, invasion, and migration of NSCLC cells through the activation of the canonical WNT/β-catenin pathway . miR-451The interactions observed between WNT/β-catenin and miRNAs are involved in the regulation of tumorigenesis in numerous cancers, such as liver cancer , colon cOn the other hand, other miRNAs appear to be tumor suppressors, such as miR-34a. miR-34a directly targets the tumor suppressor p53 and suppresses the expression of several target genes, such as SOX, Nanog, and N-Myc , 206. LeSeveral studies have shown a significant reduction of PPARγ expression in cancers such as colon cancer , 211, gaPeroxisome proliferator-activated receptor gamma agonists can induce G2/M cell cycle arrest through the stimulation of p38 MAPK in carcinoma  and in pPeroxisome proliferator-activated receptor gamma agonists can stimulate molecules involved in well-differentiated cells, such as E-cadherin, alkaline phosphatase, keratin, and carcinoembryonic antigen (CEA). This stimulation works in opposition to the non-differentiation of cells observed in cancers , 262–264Several studies have observed an anti-proliferative role played by TZDs , 265. MoThiazolidinediones downregulate tumor growth and the migration of tumor cells in colon cancer cells by inducing cell differentiation . In the The apoptotic process is stimulated by using a TZD in gastric cancer . PPARγ aPeroxisome proliferator-activated receptor gamma agonists, such as DHA and omega-3 fatty acids EPA, are known to induce anti-inflammatory activity , 253. Soin vitro and in vivo models  expression is regulated by PPARγ agonists, through a Cu/Zn-SOD promoter on the PPRE . NumerouCancers are readily associated with complex inflammatory phenomena and oxidative stress that may complicate or even initiate them. In cancers, apart from certain exceptions, the canonical WNT/β-catenin signaling is generally upregulated while PPARγ is downregulated. These two major cell pathways work in an opposite manner and this partly explains their unidirectional profile observed in cancers, chronic inflammation, and oxidative stress. This results in an activation of several upstream or downstream pathways involved in carcinogenesis, such as TGF-β, NFκB, TNF-α, TGF-β, IL-6, IL-8, VEGF, iNOS, PI3K/Akt, HIF-1α, and certain target genes such as c-Myc, cyclin D1, COX2, and Bcl2. The use of PPARγ agonists in cancers could reduce both ROS production and chronic inflammation leading to a decrease in the WNT/β-catenin pathway and then an inhibition of carcinogenesis processes Figure . BecauseAll listed authors have made substantial, direct, intellectual contributions to the study and have given their approval for its submission for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.


Fusobacterium nucleatum (Fn) in the carcinogenesis of colorectal cancer (CRC) is poorly understood. Here, we examined Fn abundance in CRC tissues, as well as β-catenin, TLR4 and PAK1 protein abundance in Fn positive and Fn negative CRCs. Furthermore, we isolated a strain of Fn (F01) from a CRC tissue and examined whether Fn (F01) infection of colon cancer cells activated β-catenin signaling via the TLR4/P-PAK1/P-β-catenin S675 cascade. Invasive Fn was abundant in 62.2% of CRC tissues. TLR4, PAK1 and nuclear β-catenin proteins were more abundant within Fn-positive over Fn-negative CRCs (P < 0.05). Fn and its lipopolysaccharide induced a significant increase in TLR4/P-PAK1/P-β-catenin S675/C-myc/CyclinD1 protein abundance, as well as in the nuclear translocation of β-catenin. Furthermore, inhibition of TLR4 or PAK1 prior to challenge with Fn significantly decreased protein abundance of P-β-catenin S675, C-myc and Cyclin D1, as well as nuclear β-catenin accumulation. Inhibition of TLR4 significantly decreased P-PAK1 protein abundance, and for the first time, we observed an interaction between TLR4 and P-PAK1 using immunoprecipitation. Our data suggest that invasive Fn activates β-catenin signaling via a TLR4/P-PAK1/P-β-catenin S675 cascade in CRC. Furthermore, TLR4 and PAK1 could be potential pharmaceutical targets for the treatment of Fn-related CRCs.The underlying mechanism of  Fusobacterium nucleatum (Fn) has been connected to an advanced disease stage and poor clinical outcome in colorectal cancer (CRC) [A growing body of evidence suggests a complex link between gut microbiota, immunity, and colorectal carcinogenesis . Recent er (CRC) , 2. HoweFn, a Gram-negative anaerobe, is associated with host immunity in colon carcinogenesis [Fn can stimulate the host immune response via toll-like receptor 4 (TLR4), which is a Gram-negative lipopolysaccharide (LPS) receptor [Recent studies reveal that ogenesis , 4. It hreceptor .It has been reported that high protein abundance of TLR4 in mouse intestinal epithelium induces colitis-associated tumor development , whereasAPC) or β-catenin genes are the main cause of β-catenin signaling activation in the majority of CRCs [It is well known that Wnt signaling pathway activation and β-catenin nuclear accumulation can be observed in approximately 90% of CRCs . Mutatio of CRCs . It was  of CRCs , 12.Fn activates β-catenin signaling through a TLR4/PAK1 cascade in CRCs. A better understanding of signaling pathways activated by Fn may offer new opportunities to target the microbiota for CRC prevention and therapy.In recent years, it has been reported that p21-activated kinase 1 (PAK1) is associated with colon cancer progression and metastasis , 14. PAKFn infection is associated with β-catenin nuclear accumulation in human CRC tissues and cells, and whether β-catenin accumulation by Fn is induced via the TLR4/PAK1 cascade. To our knowledge, this is the first study to examine the TLR4/PAK1 cascade activated by Fn in human CRC.To test this hypothesis, we examined whether Firmicutes, Proteobacteria, Bacteroidetes and Fusobacteria were the dominant phyla in most CRCs  of the CRC tissues. The clinicopathologic features of Fn-negative vs. Fn-positive lesions are shown in Table Fn-negative and Fn-positive CRCs with respect to patient gender, age, histologic grade and lymph node metastasis (P > 0.05). However, Fn was detected at a significantly higher frequency in proximal CRCs than in distal CRCs (P= 0.045).We then examined the abundance of invasive Fn-positive and 30 Fn-negative CRCs. The nuclear accumulation of β-catenin was more frequent in Fn-positive CRCs (P=0.028), suggesting that activated β-catenin signaling in these CRCs is associated with Fn infection. Similarly, TLR4 and PAK1 proteins were more abundant in Fn-positive CRCs when compared to Fn-negative CRCs (P< 0.05)  (Fn (F01) sequence has been submitted to GenBank (accession number: SUB1766768 Seq01 KX692281). SW480 and Caco-2 cells were challenged with Fn (F01) to investigate the direct influence of invasive Fn on colon cancer cells. Fn (ATCC10953) from the oral cavity and nonpathogenic E. coli were used as controls. High protein abundance of β-catenin  and its target genes, C-myc and Cyclin D1, were detected in SW480 cells . Fn (F01) challenge in SW480 cells stimulated the nuclear translocation of both total β-catenin and β-catenin S675  also stimulated the protein abundance of β-catenin S675 and its target genes C-myc and Cyclin D1  . The Fn s Figure  and Cacos Figure  infected1 Figure . Howevere Figure . These rFn activates β-catenin signaling through its LPS, we challenged SW480 cells with LPS extracted from cultured Fn (F01). LPS induced high protein abundance of P-β-catenin S675 and its target genes C-myc and Cyclin D1, with similarly increasing trends , Fn (ATCC10953) and the LPS of Fn (F01), elevations of both TLR4 and phosphor-PAK1 (P-PAK1) proteins levels began at 0.5h or 1h, and showed similar trends of growth over time  induced TLR4 increase in SW480 cells did not change as a result of pretreatment with the TLR4 inhibitor TAK-242 or the PAK1 inhibitor IPA-3. However, pretreatment of SW480 cells with TAK-242 significantly decreased P-PAK1 protein abundance, suggesting that in this instance, P-PAK1 activation could depend on TLR4. Furthermore, pretreatment with either TAK-242 or IPA-3, or both, prior to challenge with Fn (F01) led to a significant decrease of P-β-catenin S675, C-myc and Cyclin D1 with similar trends , we observed for the first time an interaction between TLR4 and P-PAK1  significantly increased the protein abundance of TLR4, P-PAK1, β-catenin S675, C-myc and Cyclin D1. Moreover, β-catenin signaling can be inhibited by both TLR4 and PAK1 inhibitors. Collectively, our results indicate that Fn may primarily activate β-catenin signaling through its LPS, subsequently involving a TLR4/PAK1 cascade in colon cancer cells.TLR4 recognizes the LPS of Gram-negative bacteria . In the Fn is a common resident of both the oral cavity and the human gut mucosa [Fn (F01) strain from a CRC patient and a Fn (ATCC10953) strain originating from the oral cavity was able to induce PAK1-mediated transcriptional activation of β-catenin. A recent study reported the abundance of some oral bacteria in the stool of patients with CRC, suggesting an oral-gut translocation route [Fn, can become opportunistic pathogens upon invasion of the gut microbiota, thereby indicating that improved oral hygiene may be useful in the prevention and treatment of CRCs. However, whether the present F01 strain, originating from CRC tissue, displays a more invasive phenotype than those originating from the oral cavity or from healthy controls, requires further investigation. Moreover, our data show that TAK-242 and IPA-3 are very effective in blocking β-catenin signaling in colon cancer cells challenged by Fn. Therefore, TLR4 and PAK1 could be putative pharmaceutical targets for the treatment of Fn-related CRCs.As a heterogeneous group of organisms, t mucosa . In the on route . Thus, sFn, we found that Porphyromonas, Parvimonas, and Gemella spp. were also abundant in some CRC tissues. Other large-scale studies have identified an over-abundance of Fn within CRC tissues. Furthermore, they have observed a significant co-occurrence of Fusobacterium, Leptotrichia and Campylobacter species [Recently it has been argued that instead of a single pathogenic bacterial species, it may be multiple species that contribute to CRC . In our  species ,29. The Fn activates β-catenin signaling via a TLR4/P-PAK1/P-β-catenin S675 cascade in the carcinogenesis of CRC; TLR4 and PAK1 could be potential pharmaceutical targets for the treatment of Fn-related CRCs.Taken together, our data suggest that invasive All tissue samples were obtained from patients undergoing colonoscopy or surgery at the Affiliated Hospital of Southwest Medical University  between February 2015 and December 2015. Fresh CRC tissue specimens (n= 14) were obtained for bacterial 16s rRNA amplification and analysis, with 7 of the samples collected from the proximal location and 7 from the distal location. The average age of the 14 CRC patients was 57.2 years. Formalin-fixed, paraffin-embedded CRC tissues (n= 98) were obtained from the pathology department archives of the same hospital for FISH analysis and immunohistochemical staining. Clinicopathologic data for each patient was obtained from the hospital records. Informed consent was obtained from all participants and the project was approved by the institutional review board.2 controlled incubator. A stock solution of the TLR4 antagonist TAK-242  was prepared in DMSO, and then further diluted with DMEM to yield a final concentration of 1 μM. TLR4 was inhibited with 1 μM TAK-242 for 1 hour prior to Fn or LPS stimulation. PAK1 was inhibited by IPA-3   for 1 hour prior to stimulation.The human colon cancer cell lines SW480 and Caco-2 were grown in DMEM high glucose, supplemented with 10% fetal bovine serum , 100 U/mL penicillin, and 0.1 mg/mL streptomycin  in a 37°C, 5% CO3 fresh CRC samples using TIANamp Bacteria DNA Kit  according to the manufacturer's protocol. The DNA was diluted to a 1 ng/μL working stock using sterile water. 16S rRNA amplification, sequencing and data analysis were performed as previously described [Total genomic DNA was extracted from 4 mmescribed . PCRs weescribed , which aPCR products were mixed in equidensity ratios. The pooled PCR products were purified with GeneJET Gel Extraction Kit . Sequencing libraries were generated using NEB Next® Ultra™ DNA Library Prep Kit for Illumina  following manufacturer's recommendations, and index codes were added. The library quantity and quality was assessed using the Qubit@ 2.0 Fluorometer  and the Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina HiSeq platform, generating 250 bp paired-end reads. Sequences were analyzed using the QIIME  softwareFn-targeted probe, FUS664, was: 5′- CTT GTA GTT CCG C(C /T) TAC CTC -3′ [Fn-positive when FUS664 probe-associated bacteria were visualized, on average, more than 10 times per field.5-μm-thick sections were prepared using samples obtained from the pathology department archives and hybridized as described in our previous study . The seq CTC -3′ . Five raIndirect immunohistochemistry was performed with formalin-fixed, paraffin-embedded tissue sections. Primary antibodies used in the study were as follows: TLR4 , PAK1 , and β-Catenin . After application of the appropriate secondary antibodies, the labeled antigens were visualized by the development of brown pigment via a standard 3,3′- diaminobenzidine (DAB) protocol. Slides were then counterstained lightly with hematoxylin. Staining without primary antibody was performed as a negative control. Immunoreactivities were estimated as described in our previous report .Total cellular protein was isolated from cultured cells with a protein extraction solution . Proteins were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes at 0.35 A  using a wet-blotting apparatus (Bio-Rad). Membranes were blocked with 5% skim milk in PBST for 2 hours at room temperature, and then incubated overnight at 4°C with the primary antibodies diluted in PBST as follows: TLR4 (1:500), PAK1 (1:1000), phosphorylated-PAK1 Thr 423 , β-Catenin (1:1000), P-β-Catenin Ser 675 , C-myc , Cyclin D1  and GAPDH . Membranes were then incubated with appropriate secondary antibodies for 1 h at room temperature. Blots were quantified by densitometry using Quantity One 4.5.0 software .Fn (F01) and a total cell lysate was obtained. SW480 cells treated with DMSO were used as a control. Total protein was immunoprecipitated using an anti-TLR4 antibody (Santa Cruz) complexed to protein A/G. The immunoprecipitated proteins were then analyzed by western blot with an antibody to detect P-PAK1 T423.The IP assay was conducted as previously described using an IP Kit  . SW480 cFn culture was performed as previously described [3 sections of proximal colon cancer tissue were thawed and immediately placed into 500 μL of sterilized PBS, washed to remove loosely associated bacteria, and then homogenized using a sterile mortar and pestle. This suspension was directly poured into fastidious anaerobe broth (FAB), supplemented with vancomycin (4 ug/mL) and neomycin sulfate (30 ug/mL), and then incubated for 1 d in a humidified anaerobic chamber . One hundred milliliter aliquots of this broth were spread onto pre-reduced brain heart infusion agar (BHI) plates supplemented with 5% defibrinated sheep blood (DSB), josamycin (3 ug/mL), vancomycin (4 ug/mL), neomycin sulfate (30 ug/mL), norfloxaxin (1 ug/mL), haemin (5 ug/mL) and menadione (1 ug/mL). Plates were inspected every 2 days for growth, and all colonies were picked and streak-purified further onto pre-reduced BHI plates supplemented with haemin (5 ug/mL), menadione (1 ug/mL), and 5% DSB. Single colonies were examined by phase microscopy, looking for slender rods or needle-shaped cells that are characteristic of Fn. DNA was extracted from positively identified isolates using TIANamp Bacteria DNA Kit. Aliquots were used as template DNA for a PCR with primers and conditions as described by Park et al [Fn. Next, verified isolates underwent a PCR to partially amplify the 16S rRNA gene for sequencing and confirmation of speciation using the same DNA template; primers and conditions are as defined by Zhao et al [Fn as the species. The isolated strain was obtained from the tumor specimen from patient number 01, and thus named F01. The strain was stored at −80°C in FAB + glycerol (50% v/v).escribed . Frozen rk et al . An amplao et al . This prFn culture was performed using an LPS extraction kit (Intron Biotechnology) according to the manufacturer's instructions.LPS extraction from the Fn  for 12 hours. Cells were then fixed in 4 % paraformaldehyde for 20 min at 4°C. After being permeabilized by incubation with 0.5 % Triton X-100 for 15 min at room temperature, cells were incubated with 0.3% hydrogen peroxide in methanol for 10 min. The slides were incubated with primary antibodies β-Catenin (1:100) and P-β-Catenin S675 (1:100) for overnight at 4°C. Glass slides were incubated with secondary antibody for 1 h at 37°C. The labelled antigens were visualized by the development of brown pigment via DAB. Slides were then counterstained lightly with hematoxylin. Staining without primary antibody was used as a negative control.The SW480 cells cultured on glass slides were treated with Three random 200× magnification fields per sample were evaluated and the average percentage of β-Catenin and P-β-Catenin S675 nuclear accumulation per field were calculated respectively.Fn (F01) was fixed with 4% glutaraldehyde overnight at 4°C, dehydrated through a graduated series of ethanol, then spread on silver paper, critical point dried and coated with gold palladium. Specimens were prepared for viewing under a JEOL-6380LV emission scanning electron microscopy .Cultured Fn (ATCC10953) was graciously supplied by Dr. Junqiang Jiang of the Affiliated Dental Hospital of Southwest Medical University, and nonpathogenic E. coli was isolated from healthy human stool for use as a control. Fn (ATCC10953) was incubated for 4 days in FAB under anaerobic conditions at 37°C. E. coli was cultured for 24 hours in Luria-Bertani broth at 37°C.Fn (F01) and Fn (ATCC10953) were cultured with FAB, supplemented with vancomycin (4 ug/mL) and neomycin sulfate (30 ug/mL) in the anaerobic system at 37°C for 4-5 days, until they reached an OD540nm of 0.8, corresponding to 109 CFU/mL. The bacteria were diluted to 107CFU/mL, corresponding to a multiplicity of infection (MOI) of 100 [Fn LPS for 1, 6, 12 and 24 hours. No bacteria or LPS were added to the control plates. The plates were shaken gently and incubated at 37°C in an incubator with controlled 5% CO2. At each time point, media was removed and cells were washed twice with PBS. Total protein was extracted from cells for western blot.The SW480 and Caco-2 cells were separately seeded in 6-well plates and incubated for 24 to 48 hours (until they reached 60% of the well) at 37°C. Cells were washed with PBS, and media was replaced with DMEM high glucose, supplemented with 10% FBS. The cells were then infected with bacteria (MOI 1:100) for 30 minutes, 1, 3, 6, 12 and 24 hours. For LPS stimulation, the SW480 cells were incubated with 0.1 ug/mL P < 0.05. All significance tests were two-tailed. All statistical tests were performed using SPSS software Version 13.0 .Data are presented as the mean and standard deviation for continuous variables and as proportions for categorical variables. Data were analyzed using one-way ANOVA, followed by Bonferroni test for multiple comparisons. Differences in categorical variables were determined by the Chi-square or Fisher's exact tests, as appropriate. Differences were considered significant if


Consequently, Dkk modulates the expression of Wnt/ β-catenin target genes. We present a mathematical model that describes the autocrine and paracrine regulation of hepatic gene expression by Dkk under wild-type conditions as well as in the presence of mutant cells.Cells are able to communicate and coordinate their function within tissues via secreted factors. Aberrant secretion by cancer cells can modulate this intercellular communication, in particular in highly organised tissues such as the liver. Hepatocytes, the major cell type of the liver, secrete Dickkopf (Dkk), which inhibits Wnt/ β-catenin signalling, and the regulation of target gene expression for 21 individual hepatocytes. Autocrine and paracrine regulation is mediated through a feedback mechanism via Dkk and Dkk diffusion along the porto-central axis. Along this axis an APC concentration gradient is modelled as experimentally detected in liver. Simulations of mutant cells demonstrate that already a single mutant cell increases overall Dkk concentration. The influence of the mutant cell on gene expression of surrounding wild-type hepatocytes is limited in magnitude and restricted to hepatocytes in close proximity. To explore the underlying molecular mechanisms, we perform a comprehensive analysis of the model parameters such as diffusion coefficient, mutation strength and feedback strength.Our spatial model describes the competition of Dkk and Wnt at receptor level, intra-cellular Wnt/ β-catenin signal transduction, allow wild-type hepatocytes to largely maintain their gene expression.Our simulations show that Dkk concentration is elevated in the presence of a mutant cell. However, the impact of these elevated Dkk levels on wild-type hepatocytes is confined in space and magnitude. The combination of inter- and intracellular processes, such as Dkk feedback, diffusion and Wnt/ The online version of this article (doi:10.1186/s12918-017-0470-9) contains supplementary material, which is available to authorized users. Cells are exposed to numerous external factors, arriving from environmental sources as well as surrounding cells. Factors secreted by cells into the extracellular space can be used for intercellular communication and by that for the coordination of cellular functions on the cell population or tissue level. Cancer cells also secrete factors but the composition and amount can differ compared to that of wild-type cells. Thereby, cancer cells can alter their microenvironment and exert a different impact on surrounding cells than wild-type cells. In consequence, gene expression and cellular functions of the surrounding cells can change. The effects are of critical importance in tissues with a complex spatial specialisation of cells, as e.g. in the liver. Here, we investigate how Dickkopf (Dkk), a factor secreted by normal and mutated hepatocytes and acting in an autocrine as well as paracrine manner, influences gene expression in the hepatic cellular environment.The liver is the largest gland of the mammalian body with a multitude of functions including blood detoxification, protein synthesis, regulation of glucose metabolism, and production of hormones and bile. It has long been known that hepatocytes, the predominant cell type of the liver, accomplish distinct biochemical tasks dependent on their location along the periportal (PP) - pericentral (PC) axis of the sinusoids of liver lobules Fig. a 1–7].–7.1–7]. β-catenin signalling pathway, in particular β-catenin and adenomatous polyposis coli (APC), are also important contributors to functional zonation [β-catenin target genes associated with ammonium metabolism such as glutamine synthetase [β-catenin or loss of Wnt co-receptor low-density-lipoprotein receptor-related proteins 5 and 6 (LRP) impair zonation emphasising the involvement of Wnt/ β-catenin signalling in its proper formation [Experimental findings indicate that intracellular components of the Wnt/ zonation –12. Immuzonation . This grnthetase . Other iormation , 11, 12.β-catenin signalling pathway is the transcriptional regulator β-catenin  family in the nucleus to regulate the expression of specific target genes [β-catenin is controlled by a multi-protein complex, referred to as destruction complex, in which APC, Axin, and glycogen synthase kinase 3 (GSK3) are central components. Bound to APC and Axin in this destruction complex, β-catenin becomes phosphorylated by GSK3 resulting in the proteasomal degradation of β-catenin [β-catenin in the cell and thus prevents the activation of Wnt/ β-catenin-dependent target genes.The central component of the Wnt/ nin Fig. b. β-Catet genes –15. The -catenin –18. Thisβ-catenin degradation [β-catenin can enter the nucleus, associate with TCF transcription factors, and regulate target gene expression [Extracellular stimulation of a cell with the morphogen Wnt directs the transmembrane receptor Frizzled (Frz) and co-receptor LRP into close proximity establishing the Wnt/Frz/LRP complex , 20. Thiradation , 24. In pression –15.β-catenin signal transduction on the receptor level such as secreted frizzled-related proteins or Dkk proteins [β-catenin signalling establishing an autocrine feedback loop [Various extracellular proteins are known to modulate Wnt/ proteins –27. In vproteins , 27. In proteins –30. By tack loop , 32. In ack loop , 34.β-catenin (19-33%) [β-catenin resulting in β-catenin accumulation even in the absence of an external Wnt stimulus. Nuclear accumulation of β-catenin has been detected in HCC samples [In hepatocellular carcinoma (HCC), a prominent type of liver cancer, Dkk is overexpressed , 36. The(19-33%) –42, Axin(19-33%) , 41, 43  samples , 42, 44 β-catenin signalling in the liver. Previously, various mathematical models have been published [β-catenin signalling pathway, such as receptor activation [β-catenin with E-cadherin [In the present study, we use a mathematical modelling approach to investigate the autocrine and paracrine effects of Dkk on the mRNA expression of target genes that are regulated by Wnt/ ublished –47 investivation –50, regutivation , 51–57, tivation –60, intecadherin , 61 or tcadherin , 62, 63.cadherin –67. Alsocadherin , 68–75. β-catenin. This reduction reflects the aberrant regulation caused by APC, Axin or β-catenin mutations that have been associated with HCC [In addition, we explore how a single mutant cell may affect gene expression in adjacent wild-type hepatocytes. We simulate this single mutant cell by a reduction of the destruction-complex-dependent degradation of with HCC –43. The The spatial model of Dkk-regulated Wnt signalling consists of a set of ordinary differential equations (ODEs) for each cell. Cells were coupled by a reaction-diffusion equation describing the evolution of Dkk, which was discretised by finite differences, yielding a set of ODEs. The equations are provided in Additional file β-catenin signalling pathway including the regulatory Dkk feedback  or can diffuse along the portal-central axis and impact the signalling of other cells (paracrine regulation). In our modelling approach, Dkk is the only component that diffuses.We aim to investigate the impact of Dkk on target gene expression in the context of an APC gradient in the liver. We focus our modelling approach on the 15-25 hepatocytes that typically align along the porto-central axis parallel to a liver sinusoid  and degraded in an APC-independent (reaction 2) and APC-dependent manner (reaction 3). The APC-dependent reaction represents the degradation of β-catenin that is mediated by the destruction complex. This reaction can be inhibited by activated Dsh (Dsh a). Dsh a is produced from inactive Dsh (Dsh i) in a Wnt/Frz/LRP receptor complex-dependent way (reaction 8) and may reconvert into Dsh i again (reaction 9). β-Catenin can reversibly form a complex with APC (reaction 4). This complex represents the binding of β-catenin to APC independent of the destruction complex [β-catenin, this complex formation acts as a positive feedback by reducing the concentration of the destruction complex [β-Catenin can also reversibly form a complex with the transcription factor TCF (reaction 5). In the model, the resulting β-catenin/TCF complex is considered to be the transcriptional activator of gene expression. TCF is produced (reaction 6) and degraded (reaction 7). The pathway components Dsh and APC obey conservation relations; that is, their respective total concentrations remain constant over time , which we consider as the readout in our model analyses. This unspecified target gene mRNA is degraded (reaction 18) but not translated for the sake of simplicity of the model. In addition to the unspecified target gene mRNA, we include the transcriptional activation of Dkk mRNA by the β-catenin/TCF complex (reaction 10). Dkk mRNA is degraded (reactions 11) or translated into Dkk. We do not explicitly consider intracellular Dkk in our model. Rather the two processes of intracellular translation of Dkk mRNA and secretion of Dkk into extracellular space are lumped into one process (reaction 12), which produces extracellular Dkk. The loss of extracellular Dkk (reaction 13) can similarly be understood as a lumped process combining clearance of Dkk from extracellular space and its intracellular degradation.We extend this signalling module by a module of target gene expression , that do not mediate Dsh i activation. The total concentrations of LRP and Frz are conserved in our model.The model furthermore integrates a previously published receptor module , which dThe model focusses on spatial effects along the porto-central axis, which is discretised into 21 segments of equal size representing the 21 hepatocytes being typically located along the axis . In the a on APC-dependent β-catenin degradation is implemented as an inhibitory function as commonly used in mathematical models of signal transduction pathways [Model parameters are collected from several experimental –80 and tpathways . The chopathways –84. DetaAutocrine and paracrine regulations are introduced in the model via Dkk feedback and Dkk diffusion. To study their effects we analyse the target gene mRNA expression in cells along the porto-central axis. Histochemical experiments in liver sections revealed that hepatic target gene expression is associated with an APC concentration gradient that increases from PC to PP region . MoreoveTo investigate the influence of the signalling processes on target gene mRNA expression, we first consider only reactions 1-9 of the signalling module and reactions 17-18 of the target gene module Fig. b. In thIn the next step, we investigate the impact of the autocrine effect of Dkk. We therefore include the Dkk feedback by taking additionally reactions 10-13 and 16 into account. To cover different possible conditions, we consider a weak or a strong feedback . The inclusion of the Dkk feedback reduces both the absolute target gene mRNA expression in each cell as well as the relative PC/PP expression ratio . This is caused by Dkk competing with Wnt for LRP binding, which reduces Wnt-induced signalling and thus target gene mRNA expression. Since the expression of Dkk per cell depends on the APC concentration, the impact of the Dkk feedback on target gene expression depends on the cell’s location within the APC gradient along the porto-central axis. The stronger the Dkk feedback, the more prominent the reduction of the target gene mRNA expression. In the case of the strong feedback , absolute target gene mRNA expression is reduced by 71% PC (cell 1) and 59% PP (cell 21) compared to the combination of signalling and target gene module . The PC/PP ratio of target gene mRNA expression is reduced from 4.5 to 3.2 .Finally, we also include the diffusion of Dkk in the extracellular space and therefore enable paracrine regulation. The assumption of a weak diffusion rate  results in a moderate change compared to the simulations without Dkk diffusion  and yields a PC/PP expression ratio of 3.8 . In contrast, higher PC/PP expression ratios are obtained by fast Dkk diffusion . In both cases the absolute target gene expression is reduced compared to the cases without Dkk regulation .−1 as reference parameters  to PP 100 nM) and constant total Wnt concentration for each cell (1 nM) Fig. a. We coion Fig. d in theion Fig. b. Such ion Fig. e. In th00 nM andIn summary, our simulations reveal that the APC gradient strongly affects the spatial profile of target gene mRNA expression. Additional gradients in Wnt might contribute to the particular shape of the profile. An opposite orientation of Wnt and APC gradients Fig. e alwaysMoreover, the setting with equal APC concentration in all cells Fig. b seems Both the scenarios in Fig. β-catenin pathway is frequently observed in human cancer such as HCC [β-catenin, Axin or APC have been detected that interfere with the degradation of β-catenin by the destruction complex [β-catenin  compared to that in a wild-type cell. The simulations show that the target gene mRNA expression of the mutant cell is strongly increased compared to that of cell 11 in the wild-type scenario  there is a limit in the impact on target gene mRNA expression. The reason is that in this extreme case, the β-catenin concentration is fixed to the ratio of its synthesis rate and APC-independent degradation rate constant . Consequently, the Dkk concentration that can be produced by a mutant cell is limited by this maximal possible β-catenin concentration . Using this assumption of maximum mutation strength, we derive an analytic equation to quantify Dkk* , which can also be experimentally determined [kp), which quantifies the maximum fraction of mutant-derived Dkk reaching wild-type hepatocytes. For any wild-type cell, we computed an upper bound for this proportion factor that only depends on the cell’s distance from the mutant cell and is independent of other model parameters Additional file 2Matlab code. (ZIP 23 kb)


Dual-Domain Carbonic Anhydrase (DDCA) had been sequenced and characterized from the ctenidia (gills) of the giant clam, Tridacna squamosa, which lives in symbiosis with zooxanthellae. DDCA was expressed predominantly in the ctenidium. The complete cDNA coding sequence of DDCA from T. squamosa comprised 1,803 bp, encoding a protein of 601 amino acids and 66.7 kDa. The deduced DDCA sequence contained two distinct α-CA domains, each with a specific catalytic site. It had a high sequence similarity with tgCA from Tridacna gigas. In T. squamosa, the DDCA was localized apically in certain epithelial cells near the base of the ctenidial filament and the epithelial cells surrounding the tertiary water channels. Due to the presence of two transmembrane regions in the DDCA, one of the Zn2+-containing active sites could be located externally and the other one inside the cell. These results denote that the ctenidial DDCA was positioned to dehydrate 2 in seawater, and to hydrate the CO2 that had permeated the apical membrane back to DDCA/DDCA in the ctenidium of T. squamosa increased significantly after 6 and 12 h of exposure to light, respectively, denoting that DDCA could participate in the light-enhanced uptake and assimilation of exogenous inorganic carbon.A  Tridacna squamosa, is one of 12 species of giant clams from the family Cardiidae and subfamily Tridacninae, which inhabit the shallow, tropical seawater of the Indo-Pacific coral reefs  from the host in order to support ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)-catalyzed photosynthesis  produced by the host is not sufficient to support the maximal rate of photosynthesis  is basically a respiratory organ with a large surface area for gas exchange, ion transport and acid-base balance in some mollusks. It is the site of light-enhanced ammonia absorption and assimilation , which are zinc-containing enzymes catalyzing these reactions with dramatic increases in the rate of CO2 hydration -like transporter which excretes H+ in exchange for Na+ and displays light-enhanced gene and protein expression . Then, Baillie and Yellowlees  was localized to the ciliated branchial filaments and cells lining the tertiary water channels in the ctenidium, in support of a role in Ci absorption. Subsequently, Leggat et al.  from its ctenidium. Particularly, efforts were made to analyze whether the DDCA comprised any transmembrane region (TM), which might shed light on the relative location of the two catalytic domains with reference to the membrane. Furthermore, the gene expression of DDCA in various tissues and organs were examined to test the hypothesis that the ctenidium was the main site of DDCA expression. Efforts were also made to determine the effects of 3, 6, or 12 h of light exposure on the transcript level of DDCA in the ctenidium, as compared with controls kept in darkness for 12 h. In order to elucidate whether the molecular changes observed, if any, were related to circadian rhythm, the effects of exposure to 12 h + 12 h  of darkness were also examined. Furthermore, a polyclonal anti-DDCA antibody was developed commercially to quantify the change in protein abundance of DDCA in the ctenidium in response to light exposure, and to elucidate the cellular and subcellular localization of DDCA in the ctenidium through immunofluorescence microscopy. It was hypothesized that the DDCA had an apical localization in the ctenidial epithelia, and its gene and protein expression levels could be up-regulated by light exposure. Results obtained were expected to provide insights into how DDCA, with its dual catalytic domains, could function to augment Ci uptake and assimilation in the ctenidium of T. squamosa during insolation.To test the hypothesis that, like No institutional  approval is required for invertebrates including giant clams at the time the laboratory experiments were performed. The animals were anaesthetized with 0.2% phenoxyethanol before killing to minimize stress and suffering.T. squamosa  were procured from Xanh Tuoi Tropical Fish, Ltd (Vietnam) and kept in an indoor aquarium. Giant clams (N = 28) were maintained in a glass tank with recirculating seawater. The water conditions and light intensity were provided as described previously . Another 12 individuals were exposed to light and sampled after 3, 6, or 12 h of light exposure (N = 4 for each time point). In order to elucidate if there was any circadian effect, four individuals were exposed to a total of 24 h of darkness, which acted as parallel controls to those exposed to 12 h of light, notwithstanding that giant clams would never encounter more than ~12 h of darkness in nature. The anaesthetized clams were dissected along the pallial line to obtain samples of the colorful and fleshy outer mantle. Thereafter, the whitish inner mantle, ctenidium, hepatopancreas, foot muscle, byssal retractor muscle, kidney and heart were quickly excised, blotted dry and immediately freeze-clamped with aluminum tongs pre-cooled by liquid nitrogen. The frozen samples were stored in −80°C until processing. For immunofluorescence microscopy, the ctenidium samples were collected from another 8 giant clams, 4 of which were exposed to darkness for 12 h and another 4 exposed to light for 12 h (N = 4 each).For molecular work, four giant clams were killed for tissue sampling at the end of a 12 h dark period, and they were regarded as controls  and the RNeasy Plus Mini Kit , respectively. A Shimadzu BioSpec-nano spectrophotometer  was used to quantify the purified RNA, and its integrity was determined by electrophoresis. Four micrograms of purified RNA were then used to synthesize the first strand cDNA using a RevertAid™ first strand cDNA synthesis kit .The extraction and purification of total RNA from the tissues of DDCA sequence was obtained using primers (Table T. gigas tgCA (AY790884.1) and Phreagena okutanii two domain membrane-associated CA (LC007965.1). The primer set was designed in such a way that they could differentiate against the two CA sequences with double domains from the algae, Dunaliella salina (U53811.1) and Porphyridium purpureum (D86051.1). PCR, cloning and sequencing procedures were performed according to the methods in Hiong et al.  and specific primers , followed by 35 cycles of 94°C (30 s), 50°C (30 s), 72°C (1 min) and 1 cycle of final extension at 72°C (10 min). Products were then separated by gel electrophoresis using a 1% agarose gel.Using specific primers Table  designedDDCA of T. squamosa was translated into the deduced amino acid sequence using the ExPASy Proteomic server . The DDCA sequence obtained was then aligned and compared with the T. gigas CA (AAX16122.1) using BioEdit . The glycosylphosphatidylinositol (GPI) anchor was predicted using the PredGPI predictor program (http://gpcr.biocomp.unibo.it/predgpi/pred.htm). A phenogramic analysis by the neighbor-joining method with 1,000 bootstrap replicates using Phylip  was not light-dependent. Using a pair of specific qPCR primers (Table -Tubulin remained unchanged throughout the 12 h of light exposure as compared to the control kept in darkness (results not shown).The mRNA expression levels of rs Table , it had Protein extraction and SDS-PAGE were performed following the methods of Hiong et al. ,b. Twent−1. Both primary and secondary antibodies were diluted in Pierce Fast Blocking Buffer (Thermo Fisher Scientific Inc.). A peptide competition assay was also performed to confirm the specificity of the anti-DDCA antibody following the methods of Hiong et al. . Results were presented as mean + S.E.M. Data were evaluated for homogeneity of variances using the Levene's test. Differences between means were evaluated using the one-way analysis of variance (ANOVA), followed by multiple comparisons of means by Tukey  or by Dunnett's T3 , and were regarded as statistically significant when DDCA (Accession number MF084997) was obtained from the ctenidium of T. squamosa. It encoded for a protein of 601 amino acids with an estimated molecular mass of 66.7 kDa. The deduced DDCA sequence shared high similarity (84.3%) with the tgCA of T. gigas (Accession number AAX16122).The complete cDNA coding sequence  of T. squamosa revealed the presence of two α-CA domains and a signal peptide  was located inside the first CA domain while the second TM (residue 580–600) was located near the 3′ end of the second CA domain adjacent to the C-terminus. The signal peptide (residue 1–22) was predicted to be extracellular, and the GPI anchor was predicted to be Ala576 located near the 3′ end of the second CA domain  that form the CO2 binding pocket, and the gatekeeper residues . In the first α-CA domain, the active binding site for + constituted five hydrophilic residues , of which His87 acted as a proton shuttle for CO2 hydration. Similarly, the Zn2+-containing catalytic site , the hydrophobic residues that form the CO2 binding pocket , and the gatekeeper residues  were conserved in the second CA domain. Unlike the first α-CA domain, six hydrophilic residues  were found in the second α-CA domain, and His363 served as a proton shuttle for CO2 hydration  origin.A phenogramic analysis indicated that the DDCA was probably a type of extracellular (membrane-bound and/or secreted) α-CA Figure . In addiDDCA was expressed predominantly in the ctenidium of T. squamosa  and the parallel control exposed to 24 h of darkness. Results from Western blotting revealed a band of interest at ~70 kDa, which was close to the estimated molecular mass of the DDCA (66.7 kDa) and therefore identified as the targeted protein , and was significantly lower than that of the clams exposed to 12 h of light  in the transcript level of s Figure . The tran Figure . There wt Figure .T. squamosa, the DDCA-immunofluoresence was detected predominantly at the apical membrane of some epithelial cells surrounding the tertiary water channels  displayed apical immunofluorescence epithelial cells as compared with the control kept in darkness for 12 h (N = 4).In s Figure  and to as Figure . Resultss Figure . In corrT. squamosa, implying that it might have a specialized function unrelated to photosynthesis of its symbionts which are concentrated in the colorful outer mantle, and unassociated with shell formation which involves the whitish inner mantle. We discovered that the DDCA from the ctenidium of T. squamosa comprised two separate Zn2+-containing active sites, one of which could be positioned extracellularly in seawater while the other one could be located intracellularly in the cytosol. In addition, the gene and protein expression levels of the DDCA in the ctenidium were enhanced by light exposure, corroborating the proposition that it may take part in increased Ci uptake from the external medium during insolation.There are indications that CAs play an important role in alga-invertebrate symbioses. Cnidarians containing endosymbiotic zooxanthellae have significantly higher CA activity (>29 times) than azooxanthellate species , and each of them contained the active site essential for CA catalytic activity as well as the histidine proton shuttle residues  in the first α-CA domain near the 5'-end, and another one (residues 580–600) near the ending of the second α-CA domain close to the 3'-end. As the two active sites happened to be separated by a TM, one of them would probably be exposed to the external medium while the other one would be positioned in the cytoplasm. A similar analysis indicated that these two TMs were also present in the tgCA of T. gigas , although no attempt had been made to identify them previously  of the first α-CA domain were located in front of the TM closer to the 5′ end, denoting that they would be exposed to the external medium. Furthermore, three of the five hydrophilic residues  of the active site which bind to +, were also located in front on the TM, while four out of the five hydrophobic residues that make up the CO2 binding pocket were positioned behind the TM. Taken together, it can be deduced for the DDCA of T. squamosa that the active site of the first α-CA domain probably functions in the external medium, binding with exogenous +, catalyzing the dehydration of 2, and releasing the CO2 to the cytoplasm. By contrast, the active site of the second α-CA domain is positioned in the cytoplasm and would therefore function intracellularly. With the 3′-end of the DDCA attached to the plasma membrane through another TM and a GPI anchor, the second α-CA domain could function in close proximity to the inner surface of the plasma membrane to catalyze the hydration of CO2 that has entered the cell to 2 and the assimilation of CO2 into T. squamosa.The CA active site is located in a large conical cavity with a catalytic ZnT. squamosa and the tgCA of T. gigas, only four other CAs are known to contain two domains. The deep-sea bivalve Phreagena okutanii  to the point that only one domain in the pair retains a functional active site  preceding the latter (at hour 12), indicating that the DDCA is regulated at both the transcriptional and translational levels. The increase in the protein abundance of the ctenidial DDCA after 12 h of light exposure was apparently not a circadian phenomenon, because such an increase was absent from the parallel controls exposed to darkness for a total of 24 h. However, whether circadian rhythm plays a role in regulating the expression levels of other genes and proteins in various tissues/organs of T. squamosa deserves a more detailed investigation in the future. Because of the daily light:dark cycle, changes in transcript levels and protein abundance of the DDCA/DDCA would logically be short-term and relatively moderate as compared with changes in other types of animal CAs in response to long-term environmental changes  of host-origin from the fleshy and colorful outer mantle of T. squamosa. CA2-like is localized to the tubule epithelial cells, and light enhances its protein abundance significantly in the outer mantle. Hence, CA2-like could probably take part in the increased supply of Ci from the host clam to the photosynthesizing symbiotic zooxanthellae during insolation. Unlike symbiotic cnidarians, giant clams have distinct tissues and organs with high degree of division of labor between them. Therefore, in the case of T. squamosa, it is unsurprising that the uptake of exogenous Ci and its delivery to the zooxanthellae by the host clam require the cooperation between the epithelial cells of the ctenidium and those of the zooxanthellal tubules with the involvement of different types of host CAs. It is probable that some other CA isoforms yet to be identified are also involved in these processes.It has been reported previously that zooxanthellae isolated from Tridacna maxima, Deane and O'Brien, T. gigas, Fitt et al., T. gigas, the concentration of Ci, present mainly as −1 (Yellowlees et al., i in the hemolymph of T. gigas decreases to 1.6 mmol l−1 (Yellowlees et al., DDCA/DDCA in the ctenidium of T. squamosa changing in response to the hemolymph T. squamosa. These include the Glutamine Synthetase (Hiong et al., 2+-ATPase (Ip et al., +/K+-ATPase α-subunit (Boo et al., T. squamosa may have developed a general mechanism to coordinate the expression levels of a variety of enzymes and transporters in relation to various diurnally light-dependent physiological processes, including light-enhanced Ci absorption, light-enhanced ammonia absorption and assimilation, and light-enhanced calcification. This could be a result of the symbiotic relationship between the host clam and the symbiotic zooxanthellae, as the host would also need to be light-responsive in order to support the light-induced photosynthesis in its symbionts.The hemolymph of giant clams has a pH of 7.4–7.6 (DDCA/DDCA and other enzymes/transporters in the host cells. Another possibility is that light is sensed through the host's siphonal eyes (Wilkens, T. squamosa.Zooxanthellae can respond to light directly probably because they possess a photoreceptor protein, opsin, and eye-spots made of crystalline clusters of uric acid (Yamashita et al., Wilkens, , which tT. squamosa would appear to be energetically uneconomical. However, unlike free-living animals, giant clams receive a continuous supply of nutrients from the symbiotic zooxanthellae, including glycerol, glucose, and amino acids (Muscatine, T. squamosa could afford energetically to regulate light-inducible processes through light-dependent diurnal changes in transcription and translation. Such diurnal changes would imply that the related transcripts and proteins must undergo rapid turnover, and therefore it would be important to determine the half-life of these proteins and to examine the regulatory mechanisms of the turnover process in the future.Transcriptional and translational processes entail energy expenditure, and therefore the diel cycle of changes in gene and protein expressions in YI designed the study and drafted the manuscript. CK, KH, CC, MB, and WW performed the experiment. CK, KH, CC, MB, SC, MN, and YI analyzed the data. WW and SC contributed reagents, materials and analysis tools. All authors reviewed 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.


The aim of our present study was to investigate the role of Wnt/β-catenin pathway in chronic TS exposure induced urocystic EMT, stemness acquisition and the preventive effect of curcumin. Long time TS exposure induced EMT changes and the levels of CSCs’ markers were significant upregulated. Furthermore, we demonstrated that Wnt/β-catenin pathway modulated TS-triggered EMT and stemness, as evidenced by the findings that TS elevated Wnt/β-catenin activation, and that TS-mediated EMT and stemness were attenuated by Wnt/β-catenin inhibition. Treatment of curcumin reversed TS-elicited activation of Wnt/β-catenin, EMT and CSCs properties. Collectively, these data indicated the regulatory role of Wnt/β-catenin in TS-triggered urocystic EMT, acquisition of CSCs properties and the chemopreventive effect of curcumin.Tobacco smoke (TS) is the most important single risk factor for bladder cancer. Epithelial–mesenchymal transition (EMT) is a transdifferentiation process, involved in the initiation of TS-related cancer. Cancer stem cells (CSCs) have an essential role in the progression of many tumors including TS-related cancer. However, the molecular mechanisms of TS exposure induced urocystic EMT and acquisition of CSCs properties remains undefined. Wnt/ Studies have shown that TS is strongly associated with the occurrence and development of bladder cancer.4 It is reported that TS is the most important single risk factor for bladder cancer with 40–60% causally related.4 Smokers have an estimated fourfold higher risk of bladder cancer than nonsmokers.5 Accumulating evidence suggested that epithelial–mesenchymal transition (EMT) and the acquisition of cancer stem cells (CSCs) properties is an important underlying mechanisms for initiation, invasion and metastasis of cancer. However, the mechanisms leading to EMT and stemness acquisition are not fully understood, which has hindered the development of effective targeted therapies and the chemoprevention of cancers including bladder cancer.Bladder cancer is the ninth most common malignancy, with an estimated 429 793 new diagnosed cases and 165 084 deaths every year worldwide.8 TS has been documented to promote EMT.11 TS-induced EMT has been found to regulate early events in cancer progression such as downregulation of epithelial cadherin, loss of cell junctions and apical–basal polarity, and enabling cells motility.11 However, the underlying mechanisms regarding how TS induces EMT and the signaling events involved remains poorly understood.EMT is a reversible naturally occurring transdifferentiation process consisting in the changes from an epithelial to a migratory mesenchymal cell phenotype. EMT has been shown to be associated with the progress of cancer, the metastatic spread and progression of cells from the site of the primary tumor to the surrounding tissues and distant organs.14 Increasing evidences have confirmed that many cancers including bladder cancer arise from a small sub-population of cancer cells, which are termed CSCs.16 CSCs with the properties of self-renewing and multipotent differentiation have an important role in human cancers progression. CSCs are also implicated in formation of metastases and relapse of malignancies. Although our previous studies showed that exposed the SV-40 immortalized human urothelial cell line (SV-HUC-1) to TS induces EMT,17 it has not been determined whether long-term TS exposure induces EMT and thereby contributes to the acquisition of CSCs properties and malignant transformation of bladder cells in vitro and in vivo.The recently uncovered link between passage through EMT and acquisition of CSCs properties indicates that the EMT programs serves as one of the mechanisms for generating CSCs.β-catenin signaling pathway is an evolutionarily conserved signaling pathway and critical for a variety of biological processes.18 Numerous studies have suggested the important functions of Wnt/β-catenin signaling in several cancers.19 In the absence of Wnt stimulation, β-catenin is phosphorylated by a destruction complex consisting of GSK3β, CK1, Axin1, Axin2 and APC, resulting in the β-catenin degradation. Upon binding of Wnt, phosphorylation of β-catenin is blocked and allows β-catenin to dissociate from the destruction complex. Then, β-catenin accumulates in cytoplasm that results in translocation of β-catenin to the nucleus and interacts with TCF/LEF to transactivate the downstream target genes including Cyclin D1, c-Myc, CD44 and ALDH etc.20 Wnt/β-catenin signaling pathway is an important inducer of EMT and critical for the maintenance of CSCs. Upon activation by specific ligands, β-catenin is released from the membrane and promotes transcription of genes involved in mesenchymal phenotype induction and the maintenance of CSCs.21 However, the role of Wnt/β-catenin in the long time TS exposure induced urocystic EMT and acquisition of CSCs properties still has not been defined.Wnt/22 Curcumin, a yellow polyphenolic compound, is the principal active component of the spice turmeric. It has long been used throughout Asia as a food additive and a traditional herbal medicine. Evidences obtained from in vitro and in vivo studies indicated that curcumin has a therapeutic potential in preventing and treating several types of cancer.23 The ability of curcumin for the target CSCs and modulation associated pathways has been proposed in several studies.25 Accumulating evidences also showed that the potent chemopreventive activity of curcumin may be partly derived from the inhibition of EMT.27Recently, some studies have illustrated that dietary phytochemicals with potent anticancer activity presented in consuming food-based dietary. It has been reported that approximately one-third of cancers can be prevented by controlling diet and regular physical activities. These effects partly have been linked to their suppressing effect on the EMT, CSCs characteristics, consequently invasion and metastasis.β-catenin pathway in chronic TS exposure induced urocystic EMT, stemness acquisition, and the preventive effect of curcumin in vitro and in vivo. Our results indicated that curcumin reverse the long-term TS exposed induced urocystic EMT, the acquisition of CSC-like properties and these effects are modulate associated with the inhibition of the Wnt/β-catenin pathway. These findings provide new insights into the pathogenesis and the chemoprevention of TS-associated bladder cancer.The aim of this study was to investigate the role of Wnt/28 To investigate the effects of cigarette smoke extract (CSE) on cell transformation, the SV-40 immortalized human urothelial cells (SV-HUC-1) were first exposed to various concentrations of CSE  for 14 days. Cells viabilities were decreased by concentrations of 2% after 14 days exposure  for about 20 weeks (48–72 h per passage). Then, we establish their doubling time and capacity for independent clone formation, the characteristics of malignant transformed cells. Colony-forming assays shown that 103±18 colonies were formed by SV-HUC-1 cells exposed to 0.5% CSE, and 182±22 colonies were formed by SV-HUC-1 cells exposed to 1% CSE. In contrast, normal SV-HUC-1 cells showed no independent clone formation . We alsoTS-induced EMT is critically involved in TS-associated malignant transformation. To evaluate the effect of chronic CSE exposure on EMT, transwell assays were carried out to analyze the migratory and invasive capacities of SV-HUC-1 cells. The results showed that CSE treatment significantly increased SV-HUC-1 cells migration and invasive capacities . To furtIn general, the EMT program tends to cause an increased expression of genes associated with 'stemness', CSCs numbers in normal or tumor cells and initiation of tumors. Whether long-term CSE exposure has a role in CSC generation need to be determined. To address this issue, a sphere formation assay was performed to evaluate the potential for self-renewal, the mRNA and protein levels of specific cell surface markers of bladder CSCs were measured. In chronic CSE exposed SV-HUC-1 cells, there was an increase in formation of spheroids . As measβ-catenin pathway. To determine whether CSE-elicited urocystic cells EMT and the acquisition of CSC-like properties were associated with changes of Wnt/β-catenin pathway, the levels of p-GSK3β, GSK3β, p-β-catenin, β-catenin, c-Myc and cyclin D1 were measured. Chronic exposure of SV-HUC-1 cells to CSE increased p-GSK3β, β-catenin, c-Myc and cyclin D1 expression levels and decreased the expression of GSK3β and p-β-catenin  was used to activate Wnt/β-catenin pathway. We showed that LiCl treatment inactivated GSK3β and increased the expression of β-catenin c-Myc and cyclin D1  of 5, 10, 15, 30 and 50 for 72 h. Green fluorescent protein (GFP) fluorescence imaging showed the successful transfection of SV-HUC-1 cells with a MOI of 30 as the optimal infection efficiency. Results showed that table transfection of GSK3β with lentiviral vector restored CSE-suppressed GSK3β activity and attenuated CSE-triggered activation of Wnt/β-catenin in SV-HUC-1 cells  for 7 days. Our results showed that CSE-induced alterations in mRNA and protein expression levels of the EMT markers, including decrease of the epithelial markers (E-cadherin and ZO-1), and increase of the mesenchymal markers (Vimentin and N-cadherin), were significantly attenuated with curcumin (10 μM) treatment ) and were exposed to TS. 30 The relationship between bladder cancer and TS has been established for TS is one of the leading causes of bladder cancer.30 However, the exact mechanisms by which TS causes the occurrence of bladder cancer remain unknown. In this study, we revealed that long time TS exposure induced bladder cells malignant transformation, EMT alterations and acquisition of stemness properties in vitro and in vivo. We further showed that chronic exposure of TS-induced bladder EMT and acquisition of CSC-like properties were associated with activation of Wnt/β-catenin signaling pathway. Meanwhile, our data indicated that curcumin attenuated TS triggered Wnt/β-catenin pathway activation and effectively prevented TS-induced urocystic EMT and acquisition of cancer CSC-like properties. These findings provide new insights into the pathogenesis and the chemoprevention of TS-associated bladder cancer.Bladder cancer is a socially significant healthcare problem and accounting for estimated 80 500 new cases diagnosed and 32 900 deaths in China. Both environmental and genetic factors are important in bladder carcinogenesis.in vitro. The viabilities of SV-HUC-1 cells exposed to CSE at concentrations of 0, 0.05, 0.1, 0.25, 0.5, 1 or 2% were examined. As shown in To investigate the mechanisms of bladder tumorigenesis induced by TS, we used CSE to mimic the effects of TS 12 Evidences have suggested that, in addition to facilitating tumor invasion and metastasis, EMT is also critically involved in the cell transformation and initiation of tumorigenesis.34 As described in this study, chronic TS exposure induced the EMT in SV-HUC-1 cells and the mice bladders, as indicated by alterations in the mRNA and protein expression levels of the EMT markers, including E-cadherin, ZO-1, Vimentin and N-cadherin. Immunofluorescent staining and immunohistochemistry also showed that long-term TS exposure decreased E-cadherin expression and increased Vimentin expression. The results suggested that TS-triggered urocystic EMT was associated with cell malignant transformation.EMT is an important process contributing to cellular transdifferentiation, the progress of cancer and the invasion/metastasis, consisting in the loss of epithelial properties and the gain of mesenchymal characteristics.35 It is reported that CD44+ bladder cancer cells represent enhanced capability of tumorigenic potential both in vitro and in vivo.36 ALDH1 is also confirmed to be one of the distinct bladder CSCs markers.35 High expression of ALDH1 was markedly associated with an advanced tumor grade, frequent tumor recurrence and poor prognosis.37 It has been reported that the pluripotent stem cell factors Nanog and Oct4 characterizing CSCs properties in many cancers including bladder cancer.39 Consistent with the previous reports, our present study shows significantly increased expression levels of these bladder CSCs markers  in chronic TS exposed SV-HUC-1 cells and the mice bladder, suggesting that these cells possess some CSCs traits.CSCs have an indispensable role in the formation, progress recurrence and metastasis of human tumors because of the highly tumorigenic, self-renewal and differentiation capabilities. CSC-like cells have been identified based on the expression of different cellular CSC markers. CD44, a specific receptor for hyaluronic acid and adhesion/homing molecule, is a common bladder CSCs surface marker.40 EMT and stemness are both extremely important characteristics for cells to acquire more invasive and metastatic potential. In this study, we also found that chronic TS exposure reinforces CSCs and EMT during the malignant transformation of bladder cells.Empirical evidence concerning the connection of EMT program to CSCs has been reported recently. As it has been reported previously, cancer cells that underwent EMT acquired characteristics of CSCs. Several lines of evidence have shown that CSCs represent a plastic state of tumor cells undergoing EMT process and exhibit a mesenchymal-like appearance.β-catenin pathway is constitutively active and promotes EMT and loss of Wnt/β-catenin pathway is associated with inhibition of CSCs stemness. In our study, we showed that Wnt/β-catenin pathway may have an important role in the long time TS exposure induced EMT and acquisition of CSC-like properties in vitro and in vivo. To determine the role of Wnt/β-catenin pathway in TS-induced EMT and acquisition of CSC-like properties, the activation effect of TS on Wnt/β-catenin was mimicked with LiCl, a specific Wnt/β-catenin pathway activator. Activation of Wnt/β-catenin alone resulted in EMT-like changes and alterations in CSCs markers expression. Furthermore, we illustrated that overexpression GSK3β by lentivirus abolished acquisition of CSC-like properties and EMT changes, indicating the essential role of Wnt/β-catenin pathway in these processes.Recent studies have focused on the signaling pathways mediated EMT and stemness. In many cancers, Wnt/46 Large amount of studies from in vitro and in vivo have indicated that curcumin exerts antioxidant, antiinflammatory, anticancer and antifibrotic properties..47 In recent years, the ability of curcumin to target CSCs has been reported in a number of in vitro studies.25 Evidence has revealed that curcumin modulate associated pathways. In this study, we found that curcumin (100mg/kg BW) inhibited the activation of Wnt/β-catenin pathway in chronic TS exposed bladder cells, as evidenced by increased GSK3β, decreased β-catenin and its downstream gene c-Myc, cyclin D1. Moreover, we demonstrated that the suppressive effects of curcumin on EMT process and changes of CSC markers expression. Taken together, these data indicated the interventional effect of curcumin on chronic TS exposure-mediated urocystic EMT and acquisition of CSC-like properties via Wnt/β-catenin inhibition.Tumor chemoprevention, that is, using natural, synthetic or biologic chemical agents to reverse, suppress or prevent the process of carcinogenesis, has been shown to be a very promising approach to prevent cancer development, especially in high-risk populations. Curcumin, a yellow coloring agent, is the principal active component of the famous spice turmeric. It has long been used for medical purposes and food additive in China and India. Curcumin is one of the most promising chemopreventive agents. Various animal and human studies have shown that curcumin is safe and excellent tolerance when administered systemically.β-catenin pathway in regulating long-term TS exposure-triggered EMT and acquisition of CSCs properties and the protective effects of curcumin on chronic TS exposure mediated urocystic EMT and acquisition of CSCs traits via Wnt/β-catenin pathway suppression. These findings may provide new insights into the molecular mechanism of TS-associated bladder tumorigenesis and the curcumin intervention.In summary, this study demonstrated the role of Wnt/See the 49 One filterless Hongtashan cigarette, one of the most consumed cigarette in China, was combusted and used for preparation of 10 ml F12K medium, which was referred to as a 100% CSE solution. SV-HUC-1 cells were exposed to various concentrations of CSE for about 40 passages.T24 cells were obtained from American Type Culture Collection  and maintained in RPMI-1640 medium. SV-HUC-1 cells were obtained from the Chinese Academy of Typical Culture Collection Cell Bank . Cells were cultured in F12K medium. CSE was prepared daily according to the reported method.To test their capacity for independent clone formation, the chronically CSE exposed SV-HUC-1 cells or control cells were plated at a density of 500 cells in 1 ml of F12K medium and medium was changed every 3 days.β or the negative control vector according to the manufacturer’s protocol.The chronically CSE exposed SV-HUC-1 cells were stably transfected with overexpression lentiviral vector for GSK3n=6). Mice were exposed to filtered air or TS with a target concentration of total particulate matter of 85 mg/m3 for 6 h per day for 12 weeks. After the last TS exposure, mice were killed and bladder tissues were collected, frozen and stored at −80 °C for further experiments.Male BALB/c mice  were purchased from the Animal Research Center of Jiangsu University. Mice were handled in accordance with the recommendations in the guidelines of Laboratory Animal Management Committee of Jiangsu University. Mice were allowed to 1-week acclimating to circumstances and then randomly assigned into each group (n=8): filtered air group; TS-exposed group, mice were exposed to TS; TS+LV-control group, mice were delivered with negative control lentiviral vector and exposed to TS; TS+LV-GSK3β group, mice were delivered with GSK3β overexpression lentiviral vector and exposed to TS. The intratracheal delivery of lentiviral vectors was performed every 4 weeks and mice were exposed to filtered air or TS for 12 weeks.In another separate animal study, mice were randomly divided into four groups (n=8): control group, mice were exposed to air and received control diet containing corn oil; TS group, mice were exposed to TS and received control diet containing corn oil; TS+Cur 50 mg/kg group, mice were exposed to TS and received diet supplemented with curcumin at dose of 50 mg/kg BW per day; TS+Cur 100 mg/kg group, mice were treated with 100 mg/kg BW curcumin and exposed to TS.Mice were treated with 50 or 100 mg/kg BW curcumin per day. Before feeding, curcumin was dissolved with corn oil. Animals were randomly assigned into each group (Four-week old BALB/c nude mice were randomly divided into two groups (four mice per group). Mouse xenograft assays were performed to detect the degree of malignancy of the chronic TS exposed cells.After chronic exposure, proteins were extracted from SV-HUC-1 cells and mouse bladder tissues. Western blot analysis were performed for the determination of target protein expression levels.qRT-PCR analyses were performed using the Power SYBR Green Master Mix by an ABI 7300 real-time PCR detection system to determine the levels of target genes. The primers used were as follows: E-cadherin forward primer 5′-TCGACACCCGATTCAAAGTGG-3′ and reverse primer 5′-TTCCAGAAACGGAGGCCTGAT-3′ ZO-1 forward primer 5′-GCAGCCACAACCAATTCATAG-3′ and reverse primer 5′-GCAGACGATGTTCATAGTTTC-3′ Vimentin forward primer 5′-CCTTGACATTGAGATTGCCA-3′ and reverse primer 5′-GTATCAACCAGAGGGAGTGA-3′ N-cadherin forward primer 5′-ATCAAGTGCCATTAGCCAAG-3′ and reverse primer 5′-CTGAGCAGTGAATGTTGTCA-3′ CD44 forward primer 5′-AGCCCATGTTGTAGCAAACC-3′ and reverse primer 5′-TGAGGTACAGGCCCTCTGAT-3′ Oct4 forward primer 5′-GTGGAGAGCAACTCCGATG-3′ and reverse primer 5′-TGCTCCAGCTTCTCCTTCTC-3′ Nanog forward primer 5′-CCTCTCCGCTTCCTTCCT-3′ and reverse primer 5′-CTGTTTGTAGCTAAGGTTCAGGAGG-3′ ALDH1 forward primer 5′-TGGCTGATTTAATCGAAAGAGAT-3′ and reverse primer 5′-TCCACCATTCATTGACTCCAPMID-3′ GAPDH forward primer 5′-GCTGCCCAACGCACCGAATA-3′ and reverse primer 5′-GAGTCAACGGATTTGGTCGT-3′.Immunofluorescent staining was performed to analyze the expression of E-cadherin, Vimentin, Nanog and OCT4 in CSE-transformed SV-HUC-1 cells.Following the completion of exposure, mice were killed and bladder tissues were collected and then the levels of E-cadherin, Vimentin, Nanog, OCT4 and ALDH1 were determined by immunohistochemistry.t-test was used. A value of P<0.05 was considered significantly different.Statistical analyses were performed with SPSS 16.0 . All data were expressed as mean±S.D. One-way ANOVA was used for comparison of statistical differences among multiple groups, followed by the LSD significant difference test. In case of comparison between two groups, unpaired Student's


Fourier transform infrared spectroscopy confirmed the existence of hydrogen bonding between the poly(ethylene oxide) segment of F127 and the OH groups of the DGEBA resin. Small-angle X-ray scattering, atomic force microscopy, and transmission electron microscopy all revealed evidence for the microphase separation of F127 within the epoxy resin. Glass transition temperature (Tg) phenomena and mechanical properties (modulus) were determined through differential scanning calorimetry and dynamic mechanical analysis, respectively, of samples at various blend compositions. The modulus data provided evidence for the formation of wormlike micelle structures, through a RIMPS mechanism, in the flexible epoxy resin upon blending with the F127 triblock copolymer.In this study, we used diglycidyl ether bisphenol A (DGEBA) as a matrix, the ABA block copolymer poly(ethylene oxide– Epoxy resins have good mechanical integrity, high thermal stability, ready processability, good chemical resistance to solvents and moisture, and exceptional adhesion to many materials. In industry, the versatility in their formulations makes epoxy resins convenient to use and widely applicable—for example, in surface coatings, painting materials, adhesives, composites, insulating materials for electrical devices, and encapsulates for semiconductors ,2. One dIn recent years, many attempts have been made to enhance the toughness of crisp epoxy resins through the self-assembly of amphiphilic block copolymers that form ordered or disordered nanostructures within thermoset polymers ,16,17,18b–propylene oxide–b–ethylene oxide) . We expected that the OH groups of diglycidyl ether bisphenol A (DGEBA) would hydrogen bond primarily with the ether groups of the PEO segment of F127 at relatively low DGEBA concentrations, but would hydrogen bond with the ether groups of both the PEO and poly(propylene oxide) (PPO) blocks at relatively higher DGEBA resin concentrations, most likely forming a series of composition-dependent nanostructures  hydrogen bonds with the PEO segment appeared at 3275 cm−1 [···ether] > [OH···OH].Gradually upon increasing the concentration of the triblock copolymer F127. Sharp peaks centered at 3373 and 3245 cm275 cm−1 , suggest−1, respectively; furthermore, the signals of the ether groups of the PEO and PPO block segments hydrogen bonded to the OH groups of DGEBA were noted at 1090 and 1082 cm−1, respectively [−1, representing the free ether groups of the PEO and PPO block segments. This peak shifted to relatively higher wavenumber upon decreasing the DGEBA concentration. When the ether groups interacted through hydrogen bonding with the OH groups of DGEBA, the electron density of the ether oxygen atoms decreased, resulting in a signal shift to lower wavenumber. The main peak of the ether shifted from 1110 to 1105 cm−1 and became sharp when the DGEBA concentration was increased. Moreover, a shoulder signal arose at 1074 cm−1 upon increasing the DGEBA concentration; this signal represented the ether groups of the PPO segment hydrogen bonded with the OH groups of the DGEBA resin.ectively . In addiTg) of the pure polymers of the PEO and PPO block segments were both approximately −62 °C; that of the epoxy resin used in this study was approximately 165 °C. Tg of PEO and PPO are very close, we could not determine the miscibility between PEO and PPO using one or two values of Tg. The one value of Tg of PEO shifted to higher values upon increasing the content of epoxy resin from 30 to 70 wt %. This value of Tg presumably arose from the epoxy/PEO phase, because the strength of inter-association between the epoxy OH groups and the PEO ether groups was greater than the strength of inter-association between the epoxy OH groups and the PPO ether groups. Tg for the epoxy resin/F127 blend; these values were higher than those predicted using the Fox rule. Next, we used TEM, SAXS, AFM, and DMA analyses to investigate the morphological transformations, self-organized structures, and mechanical properties of these immiscible epoxy/F127 blends.The samples were heated to form bulk thermoset materials of epoxy/F127 blends. DSC has been used extensively to investigate miscibility behavior in polymer blends. The glass transition temperatures (d-spacings, based on the Bragg equation. The profile of the pure triblock copolymer F127 revealed lamellar character, as determined by the specific ratio of q* and 2q* [q*, 31/2q*, and 2q* at epoxy resin contents of 30 and 40 wt %, corresponding to the long period of hexagonally packed cylinders. Further increasing the epoxy resin content led to a phase transfer with different peaks in a ratio of q* and 2q* and an increased d-spacing. Furthermore, the profile of epoxy/F127 = 70/30 featured peaks in the ratio q*, 31/2q*, and 2q*, with a decreased d-spacing, due to incomplete disordering of the hexagonal cylinders in this blend system. We recorded SAXS profiles  of the e and 2q* . We obseTg of the epoxy resin was approximately 180 °C (Tg of the PPO segment, which we could not determine in the DSC analyses, was in the approximate range from −55 to −60 °C and independent of the epoxy resin content. In addition, the samples soften increased temperature larger than Tg (epoxy resin/F127 blend from DSC, et al. investigated the impact of different nanostructures on the mechanical properties of thermoset blends with block copolymers [9 Pa when the epoxy resin content was less than 50 wt % at 25 °C. y 180 °C f; the vapolymers . The toupolymers . Figure We have prepared flexible epoxy resins through a RIMPS mechanism upon blending with the triblock copolymer F127. We have used FTIR spectroscopy, DSC, SAXS, TEM, AFM, and DMA to investigate the miscibility, self-assembly behavior, hydrogen bonding interactions, and mechanical properties of the resulting epoxy resin/F127 triblock copolymer blends. FTIR spectroscopy provided evidence that the ether groups of the PEO and PPO segments functioned as hydrogen bond acceptors for the OH groups of the DGEBA epoxy resin. The SAXS data and TEM and AFM images revealed the presence of different microphase-separated structures within the epoxy resin/F127 blends at various compositions, each mediated through hydrogen bonding interactions. DSC and DMA provided thermal and mechanical data for these blends. We observed evidence for the formation of flexible epoxy resins as a result of wormlike micelles, with a high aspect ratio and an optimal length scale, of F127 acting as the second phase, resulting in greater toughness. The modulus data provided evidence for the wormlike micelles forming, through RIMPS, flexible thermosetting materials from the epoxy resin/triblock copolymer F127 blends.


PRMT5, a protein arginine methyltransferase, has roles in cell growth regulation and cancer development. However, the role of PRMT5 in hepatocellular carcinoma (HCC) progression remains unclear. Here, we showed that PRMT5 expression was frequently upregulated in HCC tissues, and its expression was inversely correlated with overall survival in HCC patients. PRMT5 knockdown markedly inhibited in vitro HCC proliferation and in vivo tumorigenesis. We revealed that the mechanism of PRMT5‐induced proliferation was partially mediated by BTG downregulation, leading to cell cycle arrest during the G1 phase in HCC cells. Ectopic BTG2 overexpression decreased HCC growth, caused cell cycle arrest at the G1 phase, and downregulated Cyclin D1 and Cyclin E1 protein expression. Furthermore, we found that PRMT5‐induced ERK phosphorylation regulated BTG2 expression in HCC cells, whereas pretreatment with a selective ERK1/2 inhibitor (PD184352) significantly reversed the effect of PRMT5 on BTG2 expression. Our results indicated that PRMT5 promotes HCC proliferation by downregulating BTG2 expression via the ERK pathway.Increasing evidence suggests that  Hepatocellular carcinoma (HCC) is the third leading cause of cancer‐related mortality worldwide, causing approximately 600,000 deaths each year Protein arginine methyltransferase 5 (PRMT5) is the major type II arginine methyltransferase and catalyzes the symmetric methylation of histone and nonhistone proteins. Recently, accumulating evidence has shown that PRMT5 modulates cell growth and transformation. Through its methyltransferase ability, PRMT5 regulates the expression of a wide spectrum of target genes B‐cell translocation gene 2 (BTG2) was first identified in 1991 in rat PC12 cells induced by nerve growth factor and in mouse 3T3 fibroblasts stimulated with tetradecanoylphorbol acetate In this study, we found that PRMT5 was overexpressed in HCC and that PRMT5 promoted in vitro and in vivo cancer cell growth. We detected an inverse correlation between PRMT5 and BTG2 expressions. Inhibition of PRMT5 elevated BTG2 expression by activating ERK signaling. Moreover, silencing BTG2 expression reversed the effects induced by PRMT5 knockdown in HCC cells.2 at 37°C. ERK1/2 inhibitor (PD184352) and the selective PRMT5 inhibitor GSK591/EPZ015866 were purchased from Selleck .HEK 293T cells and the HCC cell lines LO2, Huh7, Bel7402, PLC/PRF/5, HepG2, MHCC97H, and SK‐Hep1 were purchased from the Chinese Academy of Sciences Cell Bank of Type Culture Collection (CBTCCCAS). The cell lines were maintained in DMEM  supplemented with 10% fetal bovine serum (Gibco) in an atmosphere of 5% COHepatocellular carcinoma and corresponding adjacent normal liver tissue samples were collected after HCC tumor resection at Sun Yat‐sen Memorial Hospital, Guangzhou. In total, 138 HCC patients who received curative resection between 2005 and 2012 at Sun Yat‐sen Memorial Hospital were enrolled in this study. The study protocol was approved by the Institute Review Board of the University of Sun Yat‐sen, and written informed consent was obtained from each patient. HCC patients with a distinctive pathologic diagnosis, no preoperative systemic or local treatment, and complete follow‐up data were selected for inclusion in this study. The pTNM classification for HCC was defined according to The American Joint Committee on Cancer/International Union Against Cancer staging system . To detect PRMT5 mRNA levels, 48 pairs of HCC samples and corresponding adjacent normal liver tissues were collected after surgical resection and stored in liquid nitrogen. A tissue microarray (TMA) consisting of 138 pairs of HCC tissues and corresponding adjacent normal liver tissues was constructed by Shanghai Biochip Company .PrimeScript RTase (Takara) according to the manufacturer's protocol. The expression levels of PRMT5 and BTG2 mRNA were determined with RT‐qPCR using Premix Ex Taq (Takara) according to the manufacturer's instructions and normalized to the expression levels of the endogenous control, GAPDH. The primers used for RT‐qPCR were as follows:Total RNA was extracted using TRIzol reagent  according to the manufacturer's instructions. Reverse transcription was performed using GAPDH: sense 5′‐GAAGGTGAAGGTCGGAGTCAACG‐3′ and antisense 5′‐TGCCATGGGTGGAATCATATTGG‐3′; PRMT5: sense 5′‐CCTGTGGAGGTGAACACAGT‐3′ and antisense 5′‐AGAGGATGGGAAACCATGAG‐3′; BTG2: sense 5′‐ATGAGCCACGGGAAGGGAA‐3′ and antisense 5′‐TTGGACGGCTTTTCGGGAA‐3′.The expression of PRMT5 and BTG2 proteins was assessed with immunohistochemistry in a tissue microarray using rabbit polyclonal antibodies specifically against PRMT5 , BTG2 , and Ki67 . Immunohistochemistry staining was performed using the Dako Envision Plus System  according to the manufacturer's protocol.Each sample was independently scored by two pathologists who were blinded to all the patient clinical data. The intensity of staining was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). Based on the percentage of positive tumor cells, the extent of staining was scored 0 (negative), 1 (1–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%). The final score for each slide was assessed by multiplying the scores for intensity and extent of staining. The slide was considered negative if the score was <5 and positive if the score was above 5.μg/mL) for 2 weeks of selection.PRMT5 (human) shRNA lentiviral vectors were purchased from Shanghai Genechem Co. Ltd. , and BTG2 (human) overexpression lentiviral vectors were purchased from Shanghai Generay Biotech Co. Ltd. Lentivirus was produced by cotransfection of 293T cells with viral vectors . Medium containing virus was collected and concentrated using PEG8000 (Sigma‐Aldrich). Infected cells were treated with puromycin  software.Post‐treatment, cells were harvested, washed twice with PBS, fixed in 75% EtOH, and stained with PI. Stained cells were analyzed with a flow cytometer using Beckman Coulter  and μm polyvinylidene difluoride membranes . Then, 5% milk in TBST was used to block nonspecific binding sites. The membranes were incubated with primary antibodies overnight at 4°C. Then, the blots were washed with 0.1% TBST three times and incubated with horseradish peroxidase‐conjugated antibodies. The protein was detected using SuperSignal West Dura . The antibodies used for western blotting were against GAPDH (sc‐293335), PRMT5 (Abcam ab109451), BTG2 (Abcam ab85051), Cyclin D1 (Abcam ab134175), Cyclin E1 (sc‐377100), p44/42 MAPK (Erk1/2) (CST #4695), phospho‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (CST #4370), phospho‐c‐Raf (Ser338) (CST #9427), Histone H4 symmetric dimethyl R3 (H4R3me2s) (Abcam ab5823), and Histone H3 (Abcam ab1791).Cells were harvested and lysed in RIPA buffer. Proteins from lysed cells were separated from 8 to 12% SDS‐PAGE gels and transferred to 0.22 Nuclear extracts were obtained with an N‐PER kit  according to the manufacturer's instructions.Small interfering RNA (siRNA) specifically targeting BTG2 and scramble siRNA  were transfected into cells using Lipofectamine 3000  according to the manufacturer's instructions.A xenograft tumor growth assay was performed by subcutaneously injecting Huh7 cells (5 × 106) stably transduced with lentiviruses encoding scrambled shRNA (shControl) or shRNA targeting PRMT5 (shPRMT5) in 0.2 mL of phosphate‐buffered saline (PBS) into the right shoulder of 6‐week‐old BALB/c nude mice. All the mice were sacrificed 4 weeks after injection. Tumors were removed and weighed. All the animal experiments were performed according to the institutional standard guidelines at Sun Yat‐sen University. The IRB number is IACUC‐DD‐17‐0803, and the animal study approved number is SYXK (Yue) 2016‐0112.t‐tests were used to compare xenograft tumor size, and PRMT5 mRNA levels and immunostaining scores in paired clinical samples. Pearson's chi‐square test was used to analyze clinical correlations. Kaplan–Meier plots and log‐rank tests were used to assess overall survival. Univariate and multivariate survival analyses were performed using Cox proportional hazards regression. Statistical results were considered significant when P < 0.05.All the statistical analyses were performed with SPSS 19.0 or GraphPad Prism 6.0. Paired n = 138). Immunostaining for PRMT5 was observed predominantly in the cytoplasm but was occasionally seen in nuclei  containing pairs of HCC specimens and corresponding pericancerous liver tissues from an HCC cohort n = 38. Immunlei Fig. .To explore the role of PRMT5 in HCC cell proliferation, we established two cell lines, Huh7‐shPRMT5 and SK‐shPRMT5, which were stably transduced with shPRMT5 lentivirus. Significant inhibition of endogenous PRMT5 expression in Huh7 and SK‐Hep1 cells was confirmed by western blotting analysis Fig. A. MTT FB and colTo determine whether PRMT5 provided an in vivo growth advantage to HCC cells, xenograft studies were performed. Tumors in mice injected with cells stably knocking down PRMT5 were smaller and lighter than those in the control group Fig. A and B. www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE56757) and prostate cancer (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE65965), after knockdown of PRMT5. However, the regulation of BTG2 by PRMT5 in HCC has not been verified. Therefore, we performed RT‐qPCR and western blotting in HCC cells after PRMT5 knockdown. Specific knockdown of PRMT5 significantly increased BTG2 mRNA levels in Huh7‐shPRMT5 cells and SK‐shPRMT5 cells by three‐ to fourfold when compared with those in the scrambled shRNA‐treated group  infected with BTG2‐overexpressing lentivirus (LV‐BTG2). BTG2 expression was confirmed by western blotting. BTG2 overexpression inhibited Cyclin D1 and Cyclin E1 expressions, as detected by immunoblotting analyses Fig. A. The grConsidering that inhibition of PRMT5 induced BTG2 expression in HCC cells and that BTG2 inhibited proliferation in HCC cells, we speculated that knockdown of PRMT5 inhibited HCC cell growth by stimulating BTG2 expression. To verify this hypothesis, Huh7‐shPRMT5 cells and SK‐shPRMT5 cells were transfected with siRNA (siBTG2), and Huh7‐shControl cells and SK‐shControl cells were transfected with scrambled RNA (siNT). The protein levels of PRMT5, BTG2, Cyclin D1, and Cyclin E1 were detected by western blotting . In addition, patients positive for PRMT5 expression exhibited shorter overall survival time and recurrence time than patients negative for PRMT5 expression. Furthermore, multivariate analysis revealed that PRMT5 expression was an independent and significant risk factor for survival and recurrence following curative resection. These data suggested that PRMT5 might be an oncogene and a useful prognostic biomarker.In this study, we explored PRMT5 oncogenicity in HCC. We found that PRMT5 was frequently upregulated in HCC tissues and HCC cell lines compared with corresponding adjacent noncancerous tissues and a normal liver cell line (LO2), respectively. PRMT5 overexpression was associated with tumor size (One of the procancer mechanisms of PRMT5 is the silencing of tumor suppressor genes. Previous studies have reported that PRMT5 regulates cell proliferation by silencing the tumor suppressor genes ST7, NM23, RBL2, and SMAD7 The MAPK pathway plays a crucial role in cell proliferation, differentiation, survival, and apoptosis Consistent with a previous report, our study showed that BTG2 upregulation significantly induced cell cycle arrest in the G1 phase and suppressed proliferation in HCC cells In conclusion, we found that PRMT5 was frequently overexpressed in HCC and that its enhanced expression was associated with a poor prognosis. PRMT5 inhibition induced cell cycle arrest in the G1 phase and inhibited in vitro and in vivo proliferation. PRMT5 inhibition increased BTG2 expression through ERK phosphorylation, thus inducing cell cycle arrest and suppressing proliferation in HCC cells. These results suggest that PRMT5 might be a potential new therapeutic target in HCC.The authors declare that they have no conflicts of interest.