As a result, serum levels of twelve total FAs (TFAs) in 1440 serum samples from 487 healthy controls (HCs), 479 patients with benign lung diseases (BLDs) and 474 patients with lung cancer (LC) were determined. Statistical analysis indicated that significantly increased levels of C16:0, C16:1, C18:0, C18:1, C18:3, C20:3, and C22:6 and decreased levels of C20:5 were observed in LC patients compared with BLDs. Receiver operating characteristic (ROC) analysis revealed that panel a , panel b , and panel c  have exhibited good diagnostic ability to differentiate BLDs from LC relative to clinical uses of tumor markers (CEA and Cyfra 21-1).In this study, we have employed graphene oxide as a matrix to simultaneously and directly quantify serum nonesterified and esterified fatty acids (FAs) using matrix-assisted laser/desorption ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS). Twelve serum nonesterified FAs combined with their individual esterified FAs ( Lung cancer (LC) is the leading cause of cancer-related death worldwide for both men and women426Fatty acids (FAs) are the major structural components of lipids that involve in many biological functions, such as energy storage, signaling molecules10121416171820222313in vivo without chromatographic separation16Traditional analysis of circulating TFAs is usually performed using gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry, both of which require complicated and time-consuming sample preparation2627i.e., C16:0, C16:1, C18:0, C18:1, C18:2, C18:3, C20:2, C20:3, C20:4, C20:5, C22:5, and C22:6) were simultaneously quantified. Statistical analysis indicated that serum levels of TFAs are closely associated with physiopathological states and that the combination of C18:2, C20:3, C20:4, C20:5, C22:5, and C22:6, the combination of C18:0, C20:4, C20:5, and C22:6, and the combination of C16:1, C18:0, C18:1, C20:3, and C22:6 have shown good diagnostic capability to differentiate BLD patients from LC patients compared with CEA and Cyfra 21-1.In this study, we have employed graphene oxide (GO) with efficient energy transfer, large thermal conductivity, and excellent electronic transportationTo establish an optimal MS condition for fragmenting phospholipids and generating serum TFAs using the QC sample as a model sample, we first optimized two important parameters: MALDI laser power and the skimmer 1 voltage of FTICR MS using GO as a MALDI matrixi.e., PA(18:1/18:1), PE(18:1/18:1), PC(18:1/18:1), PI(16:0/18:2) and PG(16:0/16:0)) were selected as model compounds. As shown in i.e., 9-AA, DMAN, and NEDC) and GO shows that GO matrix has exhibited the best performance to fragment all of the above-mentioned standard phospholipids, along with the fragmentation degree of >80%, while NEDC matrix shows the lowest degree of fragmentation for all above-mentioned standard phospholipids compared with other matrixes. Therefore, GO was selected as the MALDI matrix to analyze serum extracts.To assess the effect of different MALDI matrixes on the degree of the fragmentation of different phospholipids, several standard phospholipids  and all participants  was performed using Mann-Whitney U test. The statistical analysis indicated that there is no statistical significance between the levels of serum TFAs and gender in each physiopathological state and between three different states ; group 2, 40~49 years old (n = 157); group 3, 50~59 years old (n = 138), and group 4, 60~70 years old (n = 78)) using one-way ANOVA with LSD test after data were transformed to normal distribution. It was found that the levels of C18:2, C20:2, C20:3, C20:4, C20:5, C22:5, and C22:6 were significantly correlated with age . However, no statistical differences in the level of serum Cyfra 21-1 was observed between BLD patients and LC patients.In this study, serum tumor markers, CEA and Cyfra 21-1, were also measured in accordance with the manufacturer’s instructions and their median and ranges are listed Based on the cut-off values of serum CEA of 5.0 ng/mL, Cyfra 21-1 of 3.5 ng/mL and a combination of CEA and Cyfra 21-1 of 0.5, their AUC values were calculated to differentiate HC from BLDs, HC from LC or BLDs from LC. As shown in 16:0, C16:1, C18:0, C18:1, C18:2, C18:3, C20:4, and C22:6 were constructed with the correlation coefficients of >0.99 based on their mixture standard working solutions. Their LODs were between 0.1 μM and 2.2 μM. 144 mass spectra of the QC sample were analyzed with a relative standard deviation of 18% for all analytes. The spike-and-recovery experiments on the basis of three different concentrations of FAs indicate that their recoveries were in the range from 78.8% to 110.0%. Our data indicate that the stability and precision of this method are acceptable for complex biological sample analysis. In term of the established method, twelve TFAs from 1440 serum samples were rapidly and simultaneously quantified and the measured concentrations of serum TFAs in the present study are similar to the previously published results obtained using gas chromatograph-mass spectrometry13The detection of serum TFAs is usually performed using gas chromatography-mass spectrometry, along with a complicated and time-consuming sample preparation: lipid extraction, hydrolysis, and methylation18:2, C20:2, C20:3, C20:4, C20:5, and C22:6 in HCs and C18:3 in BLD patients  can reduce the levels of reactive oxygen species produced by macrophages, resulting in the imbalance between oxidant and antioxidant systems in LC patients20:2, C20:3, C20:4, C20:5, C22:5, and C22:6 in both BLD and LC patients are in agreement with previous studies262720:2, C20:3, C20:4, C20:5, C22:5, and C22:6 are the precursors of eicosanoid which are associated with inflammation, autoimmunity, and cancer15404220:2, C20:3, C20:4, C20:5, C22:5, and C22:6 in both BLDs and LC.Previous studies indicate that FA synthase has been found to be a hyperactivity in many cancers including LC3518:2 n-6 and C18:3 n-318:2 n-6, which affects gene expression20:418:2 in BLDs and decreased C20:4 in both BLDs and LC might be associated with changes in inflammatory response. In addition, the combinations of serum TFAs have exhibited powerful diagnostic ability to differentiate BLDs from LC with the AUC values of 0.706~0.732 relative to serum tumor markers with the AUC values of 0.521~0.588 , palmitic acid (C16:0), palmitoleic acid (C16:1), heptadecanoic acid (C17:0), stearic acid (C18:0), oleic acid (C18:1), linoleicacid (C18:2), linolenic acid (C18:3), nonadecanoic acid (C19:0), arachidonic acid (C20:4), heneicosanoic acid (C21:0), docosahexaenoic acid (C22:6), L-α-phosphatidylinositol sodium salt ), 9-aminoacridine (9-AA), 1,8-bis(dimethylamino)naphthalene (DMAN), and N-(1-naphthyl) ethylenediamine dihydrochloride (NEDC) were purchased from Sigma-Aldrich Chemicals . 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphate  (PA(18:1(9Z)/18:1(9Z))), 1,2-di-(9E-octadecenoyl)-sn-glycero-3-phosphocholine (PC(18:1(9E)/18:1(9E))), 1,2-di-(9E-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(18:1(9E)/18:1(9E))) and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol)  (PG(16:0/16:0)) were purchased from Avanti Polar lipids . GO was from Nanjing JCNANO Technology Co., Ltd . HPLC-grade isopropanol and hexane were from Fisher Scientific . Ultrapure water was purified by a Milli-Q system .Pentadecanoic acid  were excluded, and the clinical characteristics of the age-matched participants are shown in In this study, a total of 1440 overnight (more than 10 hours) fasting serum samples were collected in Peking Union Medical College Hospital . Serum samples are the remaining sera after routine physical examination or clinical examination. All samples were stored under −80 °C until use. Healthy controls (HCs) without any aberrant clinical appearance and pathomorphology were included. BLDs were diagnosed based on the clinical diagnostic criterion and LC was further confirmed by cytological or histological examination of tumor tissue. Study design is shown in Serum tumor marker levels were determined with a CEA and Cyfra 21-1 test kit  using a Cobas e601 analyzer. The cut-off values were 5.0 ng/mL for CEA and 3.5 ng/mL for Cyfra 21-1 as recommended by the manufacture’s instruction.17:0 and C21:0 were selected as internal standards (ISs), and their mixed stock solution was prepared in isopropanol at the concentrations of 375 μM C17:0 and 75 μM C21:0. The standard solutions of 6.60 mM C16:0, 1.65 mM C16:1, 10.00 mM C18:0, 10.00 mM C18:1, 10.00 mM C18:2, 0.826 mM C18:3, 3.30 mM C20:4, and 1.65 mM C22:6 were prepared in isopropanol, respectively. Then equal volume of the above-mentioned standard solutions was pooled into the mixed standard solution followed by 1-fold dilution as the stock standard solution at the final concentrations of 412.5 μM C16:0, 103.1 μM C16:1, 625.0 μM C18:0, 625.0 μM C18:1, 625.0 μM C18:2, 51.6 μM C18:3, 206.3 μM C20:4, and 103.1 μM C22:6.Cet al.17:0 and 7.5 μM C21:0), followed by the addition of 135 μL of hexane/isopropanol  and 45 μL ultrapure water. The resulting mixture was vortexed for 1 min and then stored at −20 °C overnight. After the mixture was centrifuged at 19,000 × g for 30 min at 4 °C, 20 μL of the supernatant was transferred into a new tube and air-dried. The dried sample was stored at −80 °C until use. 10 μL of isopropanol/water  was added to re-dissolve the air-dried samples for mass spectrometric analysis.Lipids were extracted from serum sample on the basis of the approach used by Hara g to remove the unexfoliated GO particles. Then the supernatant was collected for further use as a MALDI matrix. 0.3 μL of the GO solution was first pipetted on the MTP AnchorChipTM plate  and air-dried prior to the addition of 0.3 μL of the redissolved sample onto the GO matrix for mass spectrometric analysis.The GO solution (0.5 mg/mL in water) was sonicated for 2 h, followed by the centrifugation at 13,000 × TM hybrid Qh-FTICR MS  equipped with a 355 nm Nd:YAG Smartbeam II 200 Hz laser in negative ion mode. Instrument calibration was performed using a mixture of C15:0 at m/z 241.21730, C17:0 at m/z 269.24860, C19:0 at m/z 297.27990, and C21:0 at m/z 325.31120 in negative ion mode. Mass spectrum of each sample was acquired over the m/z range of 150~400 with the resolution of 200,000 at m/z 400, along with 100 laser shots per scan and the skimmer 1 voltage of −45 V in negative ion mode. The fragmentation degree of the model compounds was calculated based on the equation (1).All experiments were performed using a 9.4 T Apex-ultraMass spectral data were obtained using ApexControl 3.0.0 . After isotopic deconvolution, the resulting data were transferred to Microsoft Excel, and the half of baseline strength in each spectrum was adopted as their intensities of missing serum TFAs. Univariate analysis was performed using non-parametric Mann-Whitney U test. One-way analysis of variance (ANOVA) with Fisher’s least significant test was used to evaluate the effect of age on the levels of serum TFAs. Non-normally distributed data were transformed into normal distribution before statistical analysis. Receiver operating characteristics (ROC) curve analysis was used to calculate the area under the ROC curve (AUC), cut-off values, sensitivities, and specificities. The prediction model was further confirmed by an independent validation set based on the cut-off values obtained in the training set. Statistical analyses were performed using SPSS software . In all cases, a p value less than 0.05 was considered to be statistically significant.Serum TFAs were identified as previously describedThe reliability of MALDI-MS for quantitative analysis of FAs was validated based on the linearity, limit of detection (LOD), stability, precision, and spike-and-recovery experiments.i.e., 3.3, 6.6, 16.5, and 82.5-fold), respectively, and finally, five working standard solutions were obtained. The calibration curves between the intensity ratios of individual FAs to ISs  versus their corresponding concentration ratios were constructed based on the above-mentioned working standard solutions. C17:0 as an IS was for quantifying the levels of C16:0, C16:1, C18:0, C18:1, C18:2, and C18:3, and C21:0 as an IS was for quantifying the levels of C20:2, C20:3, C20:4, C20:5, C22:5, and C22:6. In addition, the calibration curve of C20:4 was also used for quantifying C20:2, C20:3, and C20:5 and the calibration curve of C22:6 was for quantifying C22:5 because their commercial standards are not available. Each of the working solution was analyzed three times and the results were shown as mean ± standard deviation. The LOD is defined as the concentration of each analytes at the signal-to-noise ratio of 3.To construct the calibration curves of each FA, the mixed stock standard solution was diluted to four different concentrations  serum sample obtained from the mixture of 5 HCs and 5 LC patients sera was analyzed once every 10 test samples. Finally, a total of 144 spectra of the QC sample were obtained in this study. The experimental stability and reproducibility were evaluated using the relative standard deviation (RSD) obtained based on the intensity ratios of the detected TFAs relative to their corresponding ISs. The experimental precision was assessed based on the intraday precision of three measured values of the QC sample on the same day and the interday precision of three measured values of the QC sample on three consecutive days.16:0, 2.5 μM C16:1, 15.0 μM C18:0, 15.0 μM C18:1, 15.0 μM C18:2, 1.2 μM C18:3, 5.0 μM C20:4, and 2.6 μM C22:6; R2, the concentrations of the spiked FAs: 100.0 μM C16:0, 25.0 μM C16:1, 150.0 μM C18:0, 150.0 μM C18:1, 150.0 μM C18:2, 12.0 μM C18:3, 50.0 μM C20:4, and 26.0 μM C22:6; R3, the concentration of the spiked FAs: 150.0 μM C16:0, 37.5 μM C16:1, 225.0 μM C18:0, 225.0 μM C18:1, 225.0 μM C18:2, 18.0 μM C18:3, 75.0 μM C20:4, and 39.0 μM C22:6. Lipid extraction of these three solutions were performed as described at the section of sample preparation and each was analyzed in triplicate.To assess the extraction efficiency of serum phospholipids and FAs, the spike-and-recovery experiment was employed. Briefly, 10 μL of the QC serum sample in triplicate was mixed with 10 μL ISs, 127.5 μL hexane/isopropanol , and 42.5 μL water, and then three resulting solutions were spiked with 10 μL of three different concentrations of standard FAs, respectively. Finally, three different concentration solutions of the spiked FAs are listed as below. R1, the concentrations of the spiked FAs: 10.0 μM CHow to cite this article: Ren, J. et al. Simultaneous Quantification of Serum Nonesterified and Esterified Fatty Acids as Potential Biomarkers to differentiate benign lung diseases from lung cancer. Sci. Rep.6, 34201; doi: 10.1038/srep34201 (2016).


MRTF-A gene. High expression of MRTF-A was observed in pancreatic cancer tissues and in TGF-β treated breast cancer cells. However, the mechanism for the upregulation of MRTF-A gene remains unclear. In this study, we showed that the transcription of MRTF-A was regulated by the Wnt-β-catenin signaling in breast cancer cells. LiCl treatment, Wnt3a treatment or β-catenin overexpression enhanced the transcription of MRTF-A gene. In agreement, depletion of β-catenin with siRNA diminished MRTF-A transcription. With ChIP assays, β-catenin was identified to interact with the MRTF-A promoter whereby it increased histone H4 acetylation and RNA polymerase II association. Further, results of RT-qPCR and Western-blotting supported that the transcriptional co-activator activity of MRTF-A was controlled by both the Rho-actin and the Wnt-β-catenin signaling pathways. MRTF-A was required for cell migration stimulated by the Wnt-β-catenin signaling. Taken together, our results suggest that MRTF-A integrates the Rho-actin and the Wnt-β-catenin signaling to regulate migration-related genes and consequently increases the mobility of breast cancer cells.MRTF-A is a transcriptional co-activator being critical for multiple processes including tissue fibrosis and cancer metastasis. The Rho-actin signaling stimulates the nuclear translocation and transcriptional activity of MRTF-A with little effect on the expression of  MRTF-A (myocardin-related transcription factor A), also termed MKL1, MAL and BSAC, is a transcriptional co-activator of SRF (serum response factor), which is ubiquitously expressed in a wide range of tissues –4. In nuCanis familiaris kidney epithelial (MDCK) cells, MRTF-A was shown to activate the expression of SLUG and lead to EMT  which is a process highly correlated with cancer metastasis [MRTF-A gene is upregulated in cancer cells is largely unknown.In the tastasis . It was tastasis . Recentltastasis . Thus, MMRTF-B, homologs of MRTF-A, to promote the development of lung smooth muscle [MRTF-A in breast cancer cells.In mouse lung mesenchyme, Wnt2 induced the expression of myocardin and h muscle . Althougc-MYC, SNAIL and MMPs  has been extensively studied for its roles in carcinogenesis and metastasis. The Rho-ROCK-actin signaling pathway was also well established for involvement in metastasis. Both the Rho-actin and the Wnt-β-catenin signaling pathways function in metastasis however the relationship between these pathways remains elusive.Metastasis is a complicated process regulated by multiple signaling pathways. The Wnt-β-catenin pathway which controls the expression of various oncogenes including MRTF-A was activated by the Wnt-β-catenin pathway. While the Rho-ROCK-actin signaling controlled the transcriptional activity of MRTF-A. Hence, MRTF-A integrated signals from the Rho-ROCK-actin and Wnt-β-catenin pathways to regulate migration-related genes and stimulate breast cancer cell migration.In the present study, we showed that the expression of MRTF-A, breast cancer cells MCF-7 were treated with LiCl, an inhibitor of GSK-3β which phosphorylates β-catenin for the ubiquitination-mediated protein degradation. As shown in Figure MRTF-A gene expression. To measure the expression of MRTF-A, RNA was isolated and RT-qPCR was performed. As shown in Figure MRTF-A was increased by about 2-folds following LiCl treatment, suggesting that the transcription of MRTF-A was upregulated by the Wnt-β-catenin signaling. These results were reproduced in another breast cancer cell line T47D  binding sequences near the transcription start site (TSS) of e Figure . To testMRTF-A expression, β-catenin was overexpressed and ChIP assays were performed with antibodies as indicated. Following β-catenin overexpression, the association of β-catenin became more enriched  supplemented with 10% fetal bovine serum  at 37°C in a humidified incubator with 5% COescribed , 25. A hTotal RNA was extracted with Trizol (Invitrogen). cDNA was synthesized with M-MLV reverse transcriptase (Promega) and quantified by realtime qPCR using Biosystems StepOne™ Real-Time PCR system and Fast SYBR Green Master Mix (Promega). PCR primers were designed with NCBI online software Primer-BLAST and synthesized by Invitrogen. The sequences of primers used in this study are listed in Table Proteins in whole cell lysates were separated with SDS-PAGE and then transferred to nitrocellulose membrane (Millipore). Primary antibodies including β-catenin , MRTF-A , Cyr61, MYL9  and GAPDH (Santa Cruz). Secondary antibodies were IRDye-conjugated donkey anti-mouse or anti-rabbit IgG (Licor Biosciences) and membranes were visualized with Odyssey Infrared Imaging System (Gene Company Limited).The wild type or CArG-mutated luciferase reporter plasmids were transfected into MCF-7 cells 24 h before LiCl treatmemt for another 24 h. Cells were lyzed and luciferase activities were measured with Luciferase Assay System (Promega) in a SynergyTM 4 luminometer (Bioteck).ChIP assays were carried out following the standard protocol. Briefly, MCF7 cells were cross-linked with 1% of formaldehyde (Sigma) for 15 min at room temperature with gentle shaking. Cells were harvested with cell scraper and chromatin was fragmentized to average 300 bp in length with sonicator. Diluted whole cell sonicates were incubated with anti-β-catenin , anti-RNAP II  or anti-acH4  antibodies with normal serum IgG as controls. After protein A beads  binding, washing and eluting, ChIP products were purified with phenol/chloroform and measured by real-time qPCR.Scratch wound healing assays were performed as described previously . When ce8 /L before being seeded into the upper chamber of Transwell (Corning). 0.6 ml of DMEM-F12 medium containing 10% FBS was added into the lower chamber before being cultured in an incubator. 24 h after the incubation, cells attached to the upper surface of the membrane were wiped out with cotton swabs. The membrane was fixed with 4% of paraformaldehyde and stained with DAPI to visualize the cells penetrating membrane. The number of cells was analyzed with Image J.MCF-7 or T47D cells were diluted with DMEM-F12 culture media containing 0.5% FBS to 5 × 10Cells were fixed with 4% of paraformaldehyde for 30 minutes and then permeabilized with 0.3% Triton X-100 for 15 minutes. The cells were sequentially incubated with MRTF-A antibodies and FITC-labeled secondary antibodies and washed three times with PBS and counterstained with DAPI . The cells were analyzed using a confocal laser scanning microscope . Each sample has at least three separate random fields captured from three independent experiments.t-test or one-way ANOVA analysis. All data were presented as mean ± S.E.M. p value of less than 0.05 was indicated with *, less than 0.01 was indicated with **.All statistical analyses were performed using SPSS 17.0  using either one-sample


Salmonella enterica, a well-characterized model system for activation of PhoP/Q and PmrA/B two-component systems (TCSs). In response to acidic pH, toxic metals, antimicrobial peptides, and lack of divalent cations, these TCSs modify the LPS lipid A and core, lengthen the O antigen, and upregulate specific OM proteins. An environmental change to PhoP/Q- and PmrA/B-activating conditions simultaneously induced the addition of modified species of LPS to the OM, downregulation of previously dominant species of LPS, greater OMV production, and increased OMV diameter. Comparison of the relative abundance of lipid A species present in the OM and the newly budded OMVs following two sets of rapid environmental shifts revealed the retention of lipid A species with modified phosphate moieties in the OM concomitant with the selective loss of palmitoylated species via vesiculation following exposure to moderately acidic environmental conditions.The ability of Gram-negative bacteria to carefully modulate outer membrane (OM) composition is essential to their survival. However, the asymmetric and heterogeneous structure of the Gram-negative OM poses unique challenges to the cell’s successful adaption to rapid environmental transitions. Although mechanisms to recycle and degrade OM phospholipid material exist, there is no known mechanism by which to remove unfavorable lipopolysaccharide (LPS) glycoforms, except slow dilution through cell growth. As all Gram-negative bacteria constitutively shed OM vesicles (OMVs), we propose that cells may utilize OMV formation as a way to selectively remove environmentally disadvantageous LPS species. We examined the native kinetics of OM composition during physiologically relevant environmental changes in  Salmonella enterica serovar Typhimurium following an environmental shift to PhoP/Q- and PmrA/B-inducing conditions. We show that growth conditions influence OMV production, size, and lipid A content. We further demonstrate that the lipid A content of OMVs does not fit a stochastic model of content selection, revealing the significant retention of lipid A species containing covalent modifications that mask their 1- and 4′-phosphate moieties under host-like conditions. Furthermore, palmitoylation of the lipid A to form hepta-acylated species substantially increases the likelihood of its incorporation into OMVs. These results highlight a role for the OMV response in OM remodeling and maintenance processes in Gram-negative bacteria.All Gram-negative bacteria alter the structural composition of LPS present in their OM in response to various environmental stimuli. We developed a system to track the native dynamics of lipid A change in  Gram-negative bacteria alter the composition of their outermost membrane in order to adapt to the disparate conditions encountered in various environmental niches , 2. DifffepEwzz, STwzz), LPS core decorations , and lipid A modifications   detect external conditions via inner membrane (IM) sensor kinases that can activate cognate transcription factors to promote and/or repress target gene expression . Two of R, pbgP) . These mtic cell .Certain characteristics central to the Gram-negative OM architecture and biogenesis complicate the process of rapid OM remodeling. For instance, the rate of lipopolysaccharide (LPS) diffusion in the OM is extremely low . PhosphoAll of the Gram-negative bacteria studied to date produce OM vesicles (OMVs) . OMVs arIn this study, we established a protocol in which bacteria change or maintain lipid A composition in a defined, reproducible native system. Utilizing this system, we examined OM and OMV compositions and measured OMV production levels. We compared experimentally determined relative ratios of LPS species to stochastic-model-based theoretical values in order to define the contribution of OMV production to the OM remodeling and maintenance processes of wild-type bacteria undergoing physiologically relevant changes in environmental conditions.In order to investigate the role of OMVs in the mediation and maintenance of OM remodeling, it was necessary to develop a protocol that would allow us to examine wild-type cells that are induced to accomplish a significant, measurable OM change in response to a physiologically relevant environmental shift but have a minimal opportunity to remodel their membrane via dilution upon cell growth and division. We further decided to focus on LPS since there are few known OM-localized enzymes  that are known to act on this component after it has reached the OM.Salmonella enterica serovar Typhimurium 14028S (2+ (7.6H), the lipid A subtypes present are hexa-acylated lipid A species: 1,4′-bisphosphate, 1-diphosphate, and 1-diphosphate, 4′-phosphate (2+ (5.8L), the cell’s membrane composition must rapidly adapt to the loss of magnesium ions, as the inability to bridge the acidic phosphate and pyrophosphate moieties of lipid A with divalent cations creates structural instability. Relief comes through the addition of positively charged 4-amino-4-deoxy-l-arabinose (l-Ara4N) and zwitterionic phosphoethanolamine (pEtN) groups to the phosphates of lipid A, which decrease the electrostatic repulsion between neighboring LPS molecules to stabilize OM bilayer structure  in neutral, high-Mg2+ medium 7.6H , lightly centrifuged, and gently resuspended in fresh medium under either the same condition (7.6H) or shifted to fresh, mildly acidic, low-Mg2+ medium 5.8L   in 5.8L medium, lightly centrifuged, and gently resuspended in fresh medium with either the same composition (5.8L) or a different composition (7.6H) , the 5.8L-to-5.8L mock shift in condition mimics survival and replication in an acidified SCV, the 5.8L-to-7.6H shift represents the changes upon release from an SCV, and the 7.6H-to-7.6H mock shift mimics the environment present during growth in the intestinal lumen, submucosa, or cytoplasm  . Cells en (7.6H) . The 7.6ytoplasm .Cationic antimicrobial peptide resistance arises because of the OM remodeling that occurs in response to the stimuli present under the 5.8L condition . In orde600 and CFU counts over time yielded very similar growth curves for all of the experimental conditions  and 22% phosphatidylglycerol (PG), acyl-PG, and cardiolipin (CL)  resulted in roughly comparable levels of both CL and acyl-PG between conditions. Although minor changes observed in OM-localized GPL head group composition must be considered in the comparison of cells undergoing the 7.6H-to-7.6H and 7.6H-to-5.8L shifts, GPL composition differences between the 5.8L-to-5.8L and 5.8L-to-7.6H conditions were largely indistinguishable.To further ensure comparability, the glycerophospholipid (GPL) and protein components in the OM of cells grown under each shift protocol were characterized in order to confirm that minimal changes occur during the initial 90 min of each environmental change. The pin (CL) . GPLs wepin (CL) . ExposurAnalogously, the protein compositions of OM samples purified from all of the conditions showed extensive similarity  , and one pEtN and one l-Ara4N attached at the 1- and 4′-phosphates  (see l-Ara4N lipid A species (Mass spectrometry (MS) was used to identify the lipid A species present after the 90-min shift in each condition. Three hexa-acylated lipid A species, 1,4′-bisphosphate , 1-diphosphate , and 1-diphosphate, 4′-phosphate , were confirmed to be present during the 7.6H-to-7.6H mock shift see . At 90 mp 2) see . The covp 4) see . The maj species .l-Ara4N-containing species , and a plethora of palmitoylated species  , in cultures starting from the 5.8L condition (either undergoing a mock shift [5.8L to 5.8L] or a shift [5.8L to 7.6H] in conditions), the cells have had additional time to incorporate further-modified species into the OM. Consequently, these featured many singly and doubly pEtN-containing species , a few  Da) see . Interes Da) see . The maj32P labeling and subsequent quantitative TLC analysis could not be feasibly employed. Instead, we performed densitometric analysis of the lipid A species present in the TLC bands observed by the charring method.In order to investigate the possibility that OM vesiculation could enhance OM remodeling, we needed to analyze the lipid A composition of OMVs produced explicitly over the duration of the environmental shifts. To accomplish this, large volumes of bacterial culture were necessary to obtain sufficient amounts of OMV material to yield the quantities of purified lipid A needed to perform compositional analysis of lipid A subspecies. Because of this constraint, standard methods of t = 0 min) culture, the consistency of the charring method between different plates could be further verified.For ease of quantitation, bands were labeled A to F, with partially overlapping bands grouped into a single letter category  and 1-diphosphate hexa-acylated lipid A structures  displayed identical relative intensities in the 7.6H-to-7.6H OM and OMVs  were observed in small quantities in the OM but were not detected in the OMVs. Most strikingly, lipid A species decorated with both P-l-Ara4N and P-pEtN  were seen in large quantities in the 90-min 7.6H-to-5.8L OM but were not detected in the OMVs produced over the duration of the environmental shift. These results suggest that OMVs were depleted of LPS species induced upon the shift from 7.6H to 5.8L conditions, which could indicate a positive role for OMVs in enabling OM remodeling toward an environmentally adapted LPS composition.Comparison of the lipid A composition of the final OM time point with that of the OMVs produced over the 90 min of the 7.6H-to-5.8L shift revealed major differences in the relative intensities of singly and doubly modified lipid A species (S. Typhimurium grown in Luria broth (LB), a medium depleted of divalent cations.In regard to acylation changes, the only lipid A-targeted PagP activity detected within the 90 min of the 7.6H-to-5.8L environmental shift was the palmitoylation of 1,4′-bisphosphophate lipid A  in whichOver the 90-min time period of the 5.8L-to-5.8L mock shift, the lipid A composition of the OM remained relatively static, as expected  and B. SSurprisingly, the composition of the OMVs collected from both the 5.8L-to-7.6H shift and 5.8L-to-5.8L mock shifted cells did not closely resemble the state of the OM of the cells from either condition. The 90-min 5.8L-to-5.8L OMVs were composed of 48.00% ± 1.44% B:A, 13.46% ± 0.30% C:A, 8.13% ± 7.05% D:A, 11.46% ± 4.64% E:A, 8.36% ± 4.57% F:A, 4.09% ± 0.50% G:A, and 6.50% ± 0.92% H:A, and the 5.8L-to-7.6H OMVs were composed of 34.28% ± 7.11% B:A, 13.70% ± 2.11% C:A, 13.21% ± 10.3% D:A, 16.67% ± 6.82% E:A, 10.61% ± 6.73% F:A, 6.82% ± 2.15% G:A, and 4.72% ± 3.89% H:A  and 5C. l-Ara4N-modified lipid A species  displayed comparable levels of intensity in OMVs and the OM  for the 7.6H-to-5.8L shift versus 43 ± 2.63 nm (n = 133) for the 7.6H-to-7.6H mock shift  images of OMVs. OMVs collected from 7.6H-to-7.6H mock shifted and 7.6H-to-5.8L shifted cultures  and B reck shift . Indeed,ck shift .n = 112) and 84.16 ± 2.7 nm (n = 184), respectively  to curves that modeled the progression of membrane lipid A composition. Integration of the curves representing the continuous values of each lipid A component allowed for the formulation of the theoretical composition of OMVs displayed in As the predicted lipid A composition of the 7.6H-to-7.6H mock shift OMVs closely resembled the OM compositions displayed in Intriguingly, for the 5.8L-to-5.8L condition, the theoretical model of OMV lipid A composition was vastly different from the experimentally observed composition . The B:A2+ conditions, an environment very favorable for cell growth and not generally associated with other stressors in vivo. Changing environmental conditions seem to result in delayed incorporation of newly synthesized species into OMVs. Additionally, exposure to the mildly acidic pH seems to disfavor the incorporation of the highly modified Group 4 lipid A species into OMVs, regardless of whether the cultures experienced long-term exposure to a mildly acidic pH or a recent shift from a mildly acidic pH. These deviations from the stochastic model indicate that other factors influence the regulation of OMV lipid A composition.These results indicate that the stochastic model of lipid A incorporation into OMVs seems to hold only for neutral pH/high MgThe ability of Gram-negative bacteria to rapidly alter the outermost membrane in response to changing environmental conditions is vital to their survival. In this study, we established a protocol for producing reproducible, measurable OM change in wild-type bacteria undergoing physiologically relevant changes in environmental conditions. We performed a quantitative comparison of the structural heterogeneity of the lipid A portion of LPS in the OM and OMV content produced over time.Considering the relatively low levels of LPS diffusion , 24 presIn the neutral pH, high magnesium medium, extensive cross-linking between lipid A phosphate and pyrophosphate residues creates a strong permeability barrier . The relThe 7.6H-to-5.8L environmental shift destabilizes the highly cross-linked membrane structure via the loss of divalent cations. Therefore, the progression of the OM lipid A change occurred rapidly in the environmental 7.6H-to-5.8L shift in conditions, with covalently modified species detected at the OM after only 15 min and the percentage of modified species reaching approximately 40% of the total composition after 90 min. Conversely, the modified species decreased from 80% to 50% of the total composition throughout the 90 min of the 5.8L-to-7.6H reverse shift in conditions.OMVs produced under both changing  and steady-state (5.8L to 5.8L) acidic, magnesium-limiting conditions were found to contain lesser amounts of the more highly covalently modified lipid A structures than would be predicted by modeling stochastic production based upon the OM lipid A composition over time. Likewise, the hepta-acylated 1-diphosphate, 4′-phosphate lipid A structure was found to be enriched in the OMVs secreted during the 5.8L-to-5.8L shift, while the hepta-acylated 1,4′-bisphosphate species was enriched in the OMV lipid A content during the 7.6H-to-5.8L shift. The appearance of these trends across disparate environmental shifts  points to the importance of the intrinsic physical properties of different lipid A molecules in the vesiculation process (II), are thought to aid in the formation of nonbilayer structures. Accordingly, both the geometry and associated propensity of individual membrane components to form nonbilayer lipid phases in the membrane are important to vesiculation processes  of the van der Waals volume of the base 1,4′-bisphosphate hexa-acylated lipid A lies in the hydrocarbon chains , we havencreases . Accordi−6 nm2/s   was inoculated (1:100 dilution) with bacterial cultures grown overnight . Cultures were grown to an OD600 of 0.6 and pelleted with a Beckman Avanti J-25 centrifuge , and the resulting supernatant was decanted. Cells were thoroughly resuspended in fresh N-minimal medium  and grown at 37°C for 90 min.N-minimal medium , and the resulting supernatant was filtered through a 0.45-µm cellulose membrane. The filtered supernatant was then subjected to centrifugation  before pellets were resuspended in either Dulbecco’s phosphate-buffered saline supplemented with salt  or 50 mM HEPES, pH 7.4 (for lipid A purification), and frozen at −20°C until utilization. For a large-scale OMV preparation, the cell-free supernatant was first concentrated with a tangential-flow apparatus to reduce the supernatant volume to 750 ml before centrifugation .OMVs were isolated from broth cultures as follows. One-liter cultures were pelleted with a Beckman Avanti J-25 centrifuge  pellet was boiled for 30 min in 12.5 mM sodium acetate (adjusted to pH 4.5 with acetic acid). Lipid A was then isolated via two-phase Bligh/Dyer separation and subsequent washes with fresh neutral upper phase before final drying under N2.For OMV and WC preparations, salt removal was performed by washing them three times with 50 mM HEPES, pH 7.4. We performed a variant of the protocol of Zhou et al. . The samMass spectra were acquired on a QSTAR-XL quadrupole time-of-flight tandem mass spectrometer  equipped with an ESI source. The spectra were acquired in the negative-ion mode and were largely the accumulation of 60 scans over a range of 500 to 2,500 atomic mass units . For MS 2SO4 in ethanol for detection of all species after careful, even heating at 300°C.Lipid A samples were spotted onto glass-backed silica gel 60 TLC plates and allowed to dry before development in a solvent system consisting of 25:15:4:4 chloroform-methanol-acetic acid-water. Plates were subsequently sprayed with 10% HThe lanes of each sample run on a TLC plate were set to a common width (0.29) across all plates. After selection of the lanes, the intensity of the bands was plotted by the Gel Analyzer function, with peaks inverted. The area of each peak was defined by a straight line at the base, stretching from the lowest point of the surrounding troughs, before measurement with the Wand (tracing) tool  before staining with 1% uranyl acetate .The OMVs observed on the micrographs were measured from the outermost points of the membrane with the measurement function in the NIH ImageJ software.To quantitate OMV yield, OMV preparations were boiled for 10 min in Laemmli buffer, separated by 15% SDS-PAGE, and stained overnight with SYPRO Ruby Red (Molecular Probes). The gel was fixed for 1 h in a solution of 10% methanol and 7% acetic acid before and after staining. Ruby-stained proteins were visualized under UV light. OmpF/OmpC and OmpA were quantified via densitometry (NIH ImageJ software) . A seconTable S1 Mr is the calculated molecular weight of the proposed structure; italicized values indicate less certainty in assignments.Comparison of cellular  and OMV lipid A compositions after 90-min 7.6H-to-5.8L and 5.8L-to-5.8L shifts in conditions. The Table S1, DOCX file, 0.03 MBText S1 Supplemental methods used in this study. Download Text S1, DOCX file, 0.03 MBFigure S1 Previous exposure to mildly acidic low-magnesium conditions increases survival during a PMB challenge. PMB at 0, 4, or 8 µg/ml was administered to cultures that underwent 0 (A), 45 (B), or 90 (C) min in the 7.6H-to-5.8L environmental shift protocol. These cultures were allowed to grow for 0, 2, or 4 h before CFU were counted in order to obtain the percent survival of the treated cultures. Representative results are shown. Download Figure S1, TIF file, 0.6 MBFigure S2 600 cell density and CFU measurements from 90 min under the 7.6H-to-7.6H and 7.6H-to-5.8L conditions shown on a log scale. (B) Growth curves of OD600 cell density and CFU measurements from 90 min under the 5.8L-to-5.8L and 5.8L-to-7.6H conditions shown on a log scale. (C) Relative fluorescence of cells treated with Sytox Green at different time points of environmental shift protocols compared to that of heat-killed cells under the same conditions. Download Environmental shift conditions slightly affect cell growth but not membrane integrity. (A) Growth curves of ODFigure S2, TIF file, 1.5 MBFigure S3 t = 0). (B) TEM micrograph of cells having undergone 90 min of 7.6H-to-7.6H treatment. (C) TEM micrograph of cells having undergone 90 min of 7.6H-to-5.8L treatment. (D) ImageJ quantification of the lengths and diameters of at least 25 cells chosen at random from three representative micrographs under each condition. (E to H) 2D TLC of GPLs isolated from the OM of cells having undergone 90 min of 7.6H-to-7.6H (E), 7.6H-to-5.8L (F), 5.8L-to-5.8L (G), or 5.8L-to-7.6H (H) treatment. (I) Ruby-stained SDS-PAGE analysis of the OM of cells after 90 min of environmental shift. Lanes: 1, unstained protein ladder (Bio-Rad); 2, 7.6H-to-7.6H OM; 3, 7.6H-to-5.8L OM; 4, 5.8L-to-5.8L OM; 5, 5.8L-to-7.6H OM. Download Environmental shift conditions do not affect cell morphology. (A) TEM micrograph of cells grown until directly prior to the environmental shift  TLC separation of WC lipid A from cells that have undergone the 7.6H-to-5.8L environmental shift. The bands are individually labeled on the right, and their predicted group numbers are on the far right. (B) Three WC lipid A samples are shown charred and stained with ninhydrin to identify which bands represent nitrogen-modified species (pEtN or L-4-AraN). Lanes: 1, 7.6H-to-5.8L for 75 min; 2, 7.6H-to-7.6H for 90 min; 3, 5.8L-to-5.8L for 90 min. (C) Structures of lipid A species shown with the group numbers assigned to them by H. S. Gibbons et al. . DownloaFigure S4, TIF file, 1.2 MBFigure S5 TLC densitometry band delineation and charring reproducibility. TLC separation of WC lipid A from cells that have undergone 90 min of the 7.6H-to-7.6H (A) or 7.6H-to-5.8L (B) environmental shift. In each dilution, the bands are individually labeled on the left (A to D or A to F). In panel B, two overlapping peaks are represented by the letters B and C for ease and reproducibility of quantitation. In the dotted rectangle, the gel function of ImageJ is demonstrated, with lines drawn from the troughs between the peaks of each letter grouping. Easily distinguishable noise is also excluded with lines. The area delineated from this process was measured via the wand (tracing) tool. Values were then made proportional to band A before conversion to percent composition, as shown on the right. Dark-colored columns show the values presented in Figure S5, TIF file, 1.4 MBFigure S6 Comparison of OM and OMV lipid A compositions under the 7.6H-to-7.6H, 7.6H-to-5.8L, 5.8L-to-5.8L, and 5.8L-to-7.6H conditions. The data from Figure S6, TIF file, 1.6 MBFigure S7 2+ concentration. Cells were grown overnight under four environmental conditions  before the collection of OMVs. OMV production per CFU relative to the first condition was calculated on the basis of protein content determined by OMP densitometry (D) and on the basis of lipid content determined by FM4-64 fluorescence (E). Download Evaluation of OMV density, OMV protein content, bacterial Lpp-PG cross-linking levels, and OMV production levels for growth under various environmental conditions. (A) OptiPrep density gradient fractionation of OMVs collected from 7.6H-to-7.6H and 7.6H-to-5.8L conditions was performed. Fractions were collected , run on SDS-PAGE, and stained with SYPRO Ruby Red (Molecular Probes), and OMV production levels were quantified by OMP densitometry. The level of fluorescence was normalized to the total OMP fluorescence of all of the gradient fractions. (B) Ruby-stained SDS-PAGE analysis of OMVs collected after a 90-min environmental shift. Lanes: 1, unstained protein ladder (Bio-Rad); 2, 7.6H-to-7.6H OMVs; 3, 7.6H-to-5.8L OMVs; 4, 5.8L-to-5.8L OMVs; 5, 5.8L-to-7.6H OMVs. (C) Cross-linked Lpp levels in environmentally shifted cells. Cells that underwent 90 min of the 7.6H-to-7.6H, 7.6H-to-5.8L, and 5.8L-to-5.8L environmental shifts were treated with lysozyme, PG was isolated, and the amount of Lpp copurified with the PG was analyzed by quantitative Western blotting with anti-Lpp antibody (Silhavy Lab). The amount of cross-linked Lpp was normalized to cell pellet weights, and relative amounts are shown. (D and E) Overnight OMV production under conditions varying in pH and MgFigure S7, TIF file, 1.5 MBFigure S8 Comparison of experimentally determined OMV compositions by using theoretical stochastic models. Shown are data from Figure S8, TIF file, 1.2 MB


MT1H) expression level is downregulated in several kinds of tumors, including hepatocellular cancer (HCC). However, its biological functions and underlying mechanisms in HCC is largely unknown. The current study aimed to demonstrate the expression status, biological roles and potential mechanisms of MT1H in HCC.Metallothionein 1H (MT1H in the Cancer Genome Atlas (TCGA) dataset and a panel of 12 paired tumor/non-tumor tissues. In vitro, gain-of-function experiments were performed to examine the role of MT1H on HCC cell proliferation, invasion, and migration. Using bioinformatics assay, reporter assays, quantitative real-time PCR, and western blotting, we explored the possible mechanisms underlying the role of MT1H in HCC cells. In vivo nude mice experiments were performed to assess the anti-proliferative role of MT1H in HCC.We investigated the expression level of MT1H was observed in TCGA dataset and a panel of 12 paired tumor/non-tumor tissues. Ectopic overexpression of MT1H in HepG2 and Hep3B cells inhibited cell proliferation, invasion, and migration. Gene Set Enrichment Analysis (GSEA) showed that MT1H might involve in regulation of Wnt/β-catenin pathway. Top/Fop reporter assay confirmed that MT1H had an effect on Wnt/β-catenin signaling. Real-time PCR showed MT1H expression decreased the expression of Wnt/β-catenin target genes. Western blotting assay showed that overexpression of MT1H inhibited the nuclear translocation of β-catenin and that the Akt/GSK-3β axis mediated the modulatory role of MT1H on Wnt/β-catenin signaling in HCC. In vivo nude mice experiments demonstrated that MT1H suppressed the proliferation of HCC cells. Taken together, MT1H suppressed the proliferation, invasion and migration of HCC cells via regulating Wnt/β-catenin signaling pathway.Downregulation of MT1H may be a potential target for HCC therapy.This study demonstrated that through inhibiting Wnt/β-catenin pathway, MT1H suppresses the proliferation and invasion of HCC cells. The online version of this article (doi:10.1186/s12885-017-3139-2) contains supplementary material, which is available to authorized users. Hepatocellular cancer (HCC), the third cause of cancer-related death worldwide, accounts for 85%-90% of all primary liver cancer , 2. In CMT-1 genes . MT-1 genes are reported to be involved in carcinogenesis in various human tumors [MT1F in colon cancer tissues, suggesting a tumor suppressor role for MT1F in colon cancer [MT1H in various kinds of human malignancies as compared with normal tissues, including small cell lung cancer, neuroblastoma, melanoma, B-cell lymphoma, prostate cancer, colon cancer, breast cancer, and leukemia. Furthermore, a 10- to 100-fold decrease of MT1H expression was observed in HCC in comparison with normal liver tissues, indicating a potential role of MT1H in the development and progression of HCC [The metallothioneins (MTs) are a class of cysteine-rich, low-molecular-weight, metal-binding intracellular proteins, including four identified isoforms  , 10. In n tumors . In Fu en tumors , MT1G acn cancer . Of specn cancer  found a n of HCC . NeverthThe Wnt/β-catenin signaling pathway is frequently activated during carcinogenesis, especially in HCC . In the In the current study, we identified the biological functions of MT1H in HCC and explored the possible mechanisms. Our study suggests that MT1H plays crucial role in regulating the proliferation and invasion of HCC cells through modulating Wnt/β-catenin signaling.2.Human hepatoblastoma cell lines HepG2 and Hep3B were obtained from the China Infrastructure of Cell Line Resource. The cells were cultured in Dulbecco’s modified Eagles medium (DMEM)  containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin  at 37 °C in a humidified atmosphere with 5% CODeclaration of Helsinki and was approved by the hospital’s Institutional Review Board (No. 2016397). Informed consent was obtained from each patient.Twelve HCC tissues (T) and their corresponding adjacent non-tumorous liver tissues (NT) were obtained from the surgery operation in the First Affiliated Hospital of Zhejiang University from Jan. 2015 to Dec. 2015. NT was defined as liver tissues more than 2 cm away from the edge of the tumor . Two patHuman TrueORF Gold™ pCMV6-Entry-MT1H plasmid with a C-terminal fusion of MYC/DDK tag was purchased from OriGene Technologies . To establish stable cell lines with constitutive expression of MT1H, HepG2 and Hep3B cells were transfected with pCMV6-Entry-MT1H by Lipofectamin™ 2000  according to the manufacturer’s protocol. After selection with complete medium containing G418 (0.6 mg/mL) for 2 weeks [4 cells suspended in 200 μL DMEM without FBS were added to Transwell insert  with or without coated Matrigel , respectively. The insert held in 24-well companion plates with DMEM containing 10% FBS. After 24 h incubation, the cells and Matrigel in the upper chambers were removed by cotton tip. Migrating and invading cells at the bottom of the filter were fixed, stained with 1.0% crystal violet solution and photographed under a light microscope. The number of migrating and invading cells was quantified by counting five fields in each chamber [For Transwell invasion/migration assays, the indicated 2 × 10 chamber . This exTo evaluate the cell motility, the wound-healing assay was performed. Indicated cells were seeded in 6-well plates and grown into a monolayer. In order to exclude the possible role of MT1H in proliferation that may affect the result of wound healing assay, the cells were cultured in DMEM medium supplemented with lower percentage of serum (3%). A sterile p200 pipette tip was used to make a scratch and the cells were rinsed several times with medium to remove debris and unattached cells. Cells were then cultured for 36 h and the motility of cells was photographed. Migratory activity of the indicated cells was assessed by the width of the wound, and results were quantified as the percent of control [3 cells/well. A solution of MTT  was added and cells were incubated for another 2 h at 37 °C. Then the medium was replaced by 100 μL Dimethyl sulfoxide  and shaken at room temperature for 10 min. The plate absorbance was measured at 490 nm. This experiment was repeated three separate times.HepG2-Vector, HepG2-MT1H, Hep3B-Vector and Hep3B-MT1H cells were trypsinized and seeded in 96-well plates at a density of 2 × 10Two hundred indicated cells per well were put in 12-well plates and cultured in complete medium for 10 days. The colonies were fixed with 4% paraformaldehyde and stained with 1% crystal violet solution . This exEdU incorporation assay was carried out using the Cell-Light™ EdU In Vitro kit  according to the manufacturer’s instructions. Briefly, the indicated cells added with EdU (50 μM) were cultured for 2 h, and then fixed in 4% paraformaldehyde at room temperature for 30 min. TritonX (0.5%) was used to permeabilize the cells. After incubated with 1 × Apollo reaction mixture for 30 min, the cells were subsequently stained with Hoechst 33342 for 30 min at room temperature . EdU posBriefly, the indicated cells were washed with PBS and lysed using Radioimmunoprecipitation assay buffer (RIPA)  and the amounts of protein were assayed using the Bradford method. Equal amount of protein was subjected to sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE). Proteins on the gel were transferred onto a polyvinylidene difluoride (PVDF) membrane . The membrane was blocked in 5% powdered non-fat milk in Tris-buffered saline (TBS) solution containing 0.05% Tween 20 for 1 h. Membrane was then incubated with primary antibody overnight at 4 °C, followed by incubation with secondary antibody conjugated to horseradish peroxidase at room temperature for 1 h. The signal was developed using the enhanced chemiluminescence  to expose an Xray film. The primary antibodies against Flag, β-catenin, lamin a, α-Tubulin , phospho-Akt (Ser473), Akt, phospho-GSK-3β (Ser9), and GSK-3β , were used. The experiment was repeated three times to confirm the results. The intensity of the indicated bands was quantified using Quantity One software .Indicated cells were grown on coverslip  on 24-well plate were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X in PBS. β-catenin were detected by incubation with specific anti-β-catenin antibody  overnight at 4 °C. A secondary antibody (donkey anti-rabbit rhodamine red)  was used before staining the nuclei with 1 μg/mL DAPI . Pictures were acquired by using a fluorescence microscope .Total RNA was extracted from the cells or tissues using Trizol reagent , and reverse transcription was conducted as follows with random primers and Moloney murine leukemia virus reverse transcriptase  in accordance with the manufacturer’s protocol. The original amount of the specific transcripts were measured by real-time PCR according to a standard protocol with a SYBR Green PCR kit  using the ABI Prism 7000 sequence detection system . For each gene and sample, three independent assays were conducted and the comparative Ct method was performed to calculate the relative abundance of mRNA compared with that of the endogenous reference control β-actin. The primers used were listed in Table Nuclear protein extraction was carried out using Active Motif Nuclear Extraction Kit  according to the manufacture’s protocol. Lamin A was used as nuclear fraction controls .The Top/Fop flash plasmid system  was used to evaluate the transcriptional activity of TCF. Topflash and Fopflash plasmids were co-transfected in the indicated cells using Lipofectamin™ 2000 . The luciferase values were measured after 48 h incubation, using Dual-Luciferase reporter assay system  according to the manufacturer’s instructions . The exp6 cells) were injected subcutaneously into the right flanks of female BALB/c athymic nude mice (4–6 weeks of age), five mice per group [2). At 30 days after injection, all mice were sacrificed and tumors were weighted. This experiment was performed in accordance with the institutional ethical guidelines for animal experiment.HepG2-Vector and HepG2-MT1H (1.5 × 10er group . The tumhttp://software.broadinstitute.org/gsea/index.jsp) was performed as previously described [http://software.broadinstitute.org/gsea/msigdb/cards/WNT_SIGNALING.html) were downloaded from GSEA and the genes are listed in Additional file GSEA . Continuous variables were presented as mean ± SD and analyzed using the Student’s t-test. The relationship between MT1H expression in large datasets derived from TCGA databases [MT1H is significantly downregulated in most (48/50) HCCs . By using TCGA dataset and the GSEA assay, we found that Wnt/β-catenin pathway related genes were significantly enriched in the HCC patients with low MT1H expression  expression can be found in TCGA data. We categorized the Wnt/β-catenin target genes’ expression into two groups: low expression group  and high expression group . As shown in Additional file POU5F1, DKK1, NOS2, MSL1, MYCBP, NRCAM, LEF1, and PPARD transcripts were found in HCC tissues versus adjacent non-tumorous liver tissues. Taken together, these data suggest that MT1H plays a potential role in regulating β-catenin signaling in HCC.Next, we sought to explore the driving mechanisms behind the effects of MT1H on HCC cell proliferation, invasion as well as migration. We used LIHC RNAseq2 data generated by the TCGA and sorted the samples into the top and bottom quartiles of ion Fig. . MoreoveMT1H in HepG2 and Hep3B cells significantly suppressed the levels of MYC, MMP7, and LEF1  [MT1H in HCC need to be further explored. Delineating the precise molecular mechanisms for the altered expression of MT1H in HCC will put new insights into the understanding of HCC tumorigenesis.The mechanisms underlying the downregulation of MTs have been investigated in malignant diseases , 33–40. ty (LOH) . In addity (LOH) . WhetherGiven that Wnt/β-catenin pathway plays an essential role in tumorigenesis, together with our bioinformatics analysis implicated that Wnt/β-catenin signaling might involve in the function of MT1H in HCC. It is important to explore the tumor suppressor role of MT1H mediated by Wnt/β-catenin signaling in HCC. We found that MT1H suppressed β-catenin nuclear translocation and transcriptional activity in HCC. Wnt/β-catenin signaling has demonstrated to have important effects on proliferation and invasion of tumor cells. The activated Wnt/β-catenin pathway has been observed in at least one-third HCCs and most of them have mutations in the β-catenin gene . TherefoAkt can lead to phosphorylated inactivation of GSK-3β, which in turn results in β-catenin stabilization and nuclear transportation . Of noteMT1H and the interaction between MT1H and Akt/GSK-3β/β-catenin signaling.In summary, our data showed that through inhibiting Wnt/β-catenin pathway, MT1H could suppress the proliferation and invasion of HCC cells. MT1H might be a potential new target for HCC therapy. Ongoing studies are to explore the molecular mechanism of downregulated


This is accompanied by actin cytoskeleton reorganization with increased cortical actin deposition and downregulation of known Wnt target genes that are associated with epithelial to mesenchymal transition (EMT) and cancer cell invasion. Wnt pathway inhibition by SID decoy peptide was confirmed by decreased Wnt reporter activity and altered cytoplasmic localization of nuclear β-catenin. TGIF1, a transcription factor that modulates Wnt signaling and known to interact with the PAH2 domain of SIN3A, can be dissociated from the SIN3A complex by SID decoys. TGIF1 knockdown inhibits WNT target genes and in vitro cell invasion suggesting that TGIF1 might be a key target of the SID decoys to block tumor invasion. Taken together, targeting SIN3 function using SID decoys is a novel strategy to reverse invasion and the EMT program in TNBC translating into the inhibition of metastasis dissemination and eradication of residual disease.Cancer cell invasion is an obligatory step for metastatic dissemination that contributes to rapid relapse and a poorer survival in triple negative breast cancer (TNBC) patients. Development of novel therapeutic strategies to block tumor invasion is an unmet need in the treatment of cancer. We reported that the selective inhibition of the PAH2 domain of SIN3A protein function markedly suppressed metastatic dissemination to the lungs in TNBC xenograft bearing mice. Here, we show that TNBC cell lines treated with Sin3 interaction domain (SID) decoy peptides that bind to PAH2 display a strong  Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with high incidence in young African American women (∼30 years old), or patients with BRCA1/BRCA2 mutation . The lacin vitro or in vivo with SID decoys promotes differentiation and luminal phenotype coincident with inhibition of epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and distant metastasis [Our previous work has established the role of Sin3 paralogues (SIN3A and SIN3B) in TNBC pathogenesis –9. Withotastasis , 12.Underlying these strong anti-tumor effects, SID decoys inhibit key oncogenic signaling including the Wnt pathway. Wnt signaling is hyperactivated in 40-60% of TNBCs through strong expression of nuclear β-catenin and its targets , 14 and Here we report that SID decoy mediated inhibition of invasion and Wnt/β-catenin signaling of TNBC can result from the disruption of protein-protein interaction between the PAH2 domain of SIN3A and SID containing transcription factor TGIF1. Our novel findings identify SIN3A-TGIF1 protein interaction as a potential therapeutic target for preventing and treating metastases in patients with TNBC.We have previously shown that stable expression of SID peptide markedly inhibits growth of invasive TNBC colonies in 3-demensional (3D) Matrigel™ , 12. ConSince cancer cell invasion and migration require dynamic cytoskeletal reorganization , 21, we Pathway analysis of expression microarray data obtained from the MDA-MB-231 cells treated with 2.5μM SID peptide for 24 hours  identifiCTNNB1, LEF1, MMP9, MT1-MMP, PLAU) in MDA-MB-231 cells treated with SID peptide. Treatment with SID peptide significantly decreased the expression of all the genes tested by qRT-PCR   Figure . Functio) Figure . Western) Figure , S4A-S4B) Figure .-15)   occupancy in a cluster of gene sets . We exam) Figure . Taken tSince only nuclear β-catenin co-opts with Wnt signaling , we alsoWe next questioned if the inhibition of invasion and Wnt signaling by SID peptide is due to disruption of protein interactions specific to the PAH2 domain of SIN3A. Of the limited number of proteins known to interact with PAH2 domain , the SIDTo further investigate the role of SIN3A-TGIF1 interaction in invasion and Wnt signaling, TGIF1 protein was depleted in D3H2LN cells by transfecting a pool of three TGIF1-targeting siRNAs (siTGIF1) Figure . A markein vitro cultured tumor cell lines and patient samples led us to identify novel genetic and epigenetic aberrations that contribute to tumor progression, which could be utilized for the development of novel targeted therapies [in vivo in TNBC xenograft mouse models with minimum cytotoxicity [Therapeutic intervention for TNBC is hampered due to tumor cell heterogeneity and lack of major molecular targets . Recent herapies , 28. Preherapies . We repotoxicity , 11. In toxicity –12.Here, we show that by blocking the interaction of SIN3A with selective partners (transcription factors and chromatin modifiers) through its PAH2 domain using SID peptide, the invasive phenotype of TNBC cells is suppressed and that this process is mediated by the impairment of Wnt signaling. We anticipated that SID peptide might also modulate Wnt pathway since ivermectin, shown previously to have a SID decoy mimetic activity , also suOur results show that SID peptide attenuates Wnt/β-catenin pathway activity, at least in part, by promoting the translocation of nuclear β-catenin into the cytoplasm Figure  that leaOur data and others indicate that the SIN3A or Sin3B/HDAC complex(es), generally known as a part of transcriptional repressor complexes , can medTreatment with SID peptide for 24 hours downregulates Wnt pathway targets and EMT-related genes and suppresses invasion in TNBC cells. We found that SIN3A in complex with TGIF1 promotes TNBC invasion and Wnt/β-catenin pathway, which is inhibited by SID decoys by attenuating SIN3A-TGIF1 interaction. E-cadherin is induced 72 hours after SID peptide treatment in TNBC , which iin vivo with minimum cytotoxicity [in vivo to explore their potential use in TNBC patients.We have shown that SID decoys could be a novel anti-metastatic therapy for TNBC by selectively targeting TGIF1, one of the SID containing transcription factors. Targeted inhibition of TGIF1 by SID decoys leads to the inhibition of hyperactive Wnt pathway which occurs in about 40-60 % of TNBC cases , 14, 17.toxicity –12, whicThe human MDA-MB-231 breast adenocarcinoma (Cat# HTB-26) and MDA-MB-157 breast medullary carcinoma (Cat# HTB-24) cell lines were purchased from the American Type Culture Collection (ATCC). The MDA-MB-231-Luc-D3H2LN Bioware (D3H2LN) cell line was purchased from PerkinElmer (Cat# 119369). Mouse 4T1 cells (Cat# CRL-2539) were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) and 1% Antibiotic-Antimycotic solution (Invitrogen). D3H2LN, MDA-MB-231, and MDA-MB-157 cell lines were maintained in DMEM supplemented with 10% FBS, 1% GlutaMAX (Invitrogen), 10mM HEPES, 1mM sodium pyruvate, non-essential amino acids and 1% antibiotic-antimycotic solution.YGRKKRRQGGG-VRMNIQMLLEAADYLERRER). SCR peptide in addition to TAT peptide has the following sequence: YGRKKRRQGGG-EQRARRIMERLLEYNMVADL.SID and scramble (SCR) peptides were generated as previously described , 12. SIDImmunoblotting was performed according to the standard procedures and visualized using ECL plus Western Blotting detection reagent (Life Technologies). Blots were probed with anti-β-catenin rabbit mAb , SIN3A (AK-11) antibody , TGIF1 antibody , and anti-GAPDH antibody . Immunoprecipitation was performed according to the standard procedures.4) were seeded onto Matrigel-coated a 24-well filter insert (Corning) and 5% FBS was used as chemoattractant in the lower chamber. The numbers of invading cells were counted after 24 hours, stained with HEMA 3 stain set (Fisher Scientific), and the number of invasive cells after treatment was normalized to that of untreated cells (UT) and calculated as the percentage of invasive cells. The total 5 fields were counted per filter under 200x magnification using a Nikon Eclipse E600 microscope. For transwell migration assay, cells were seeded onto the top well of a 24-well filter insert without Matrigel coating and the same procedures as invasion assays were performed afterwards.TNBC cells (5x10D3H2LN or MDA-MB-157 cells were plated onto 8 well chamber coated with thin layer of Matrigel diluted in PBS (1:100 dilution) and treated with SCR or SID peptide (2.5μM) for 24 hours. Cells were stained with rabbit β-catenin (D10A8) mAb , TCF7L2 , rabbit LEF1 (C12A5)  or rabbit polyclonal TGIF1 (H-172) antibody  1:100-1:250 overnight at 4°C according to standard protocols. Cells were fixed with 4% paraformaldehyde at RT for 15 min and incubated with 0.5% Triton-X-100 at 37°C for 5 min, and blocked with 5% bovine serum albumin at RT for 1 hour. Secondary antibodies  were added for 1 h and then washed with PBS. Cells were mounted in Prolong Gold antifade reagent  Images were collected on a Leica SP5 confocal microscope.The number of cells with “nuclear”, “nuclear/cytoplasmic”, or “cytoplasmic” β-catenin was quantified from immunofluorescence images (6-9 fields quantified per sample). For quantification, we used plugins -> particle analysis-> cell counter menu in ImageJ program (NIH). Cells that have fluorescence intensity above designated threshold level in ImageJ were considered as β-catenin positive cells. The number of “nuclear”, “nuclear/cytoplasmic”, or “cytoplasmic” β-catenin positive cells was normalized to the total number of cells counted from multiple fields and converted into the percentage of β-catenin positive cells. The cells with “nuclear β-catenin” staining demonstrate those with β-catenin localization exclusively in the nucleus. The cells with “nuclear/cytoplasmic β-catenin” show distribution of nuclear localization of β-catenin that are weaker than cells with “nuclear β-catenin” and have some cytoplasmic distribution. The cells with “cytoplasmic β-catenin” show ubiquitous localization of β-catenin staining without strong fluorescence signal concentrated in either nucleus or cytoplasm.4 cells were plated per well on 24-well plates and transduced with Top flash and Fop flash supernatant. The next day, cells were treated with 2.5μM SID or SCR peptide in regular media containing 5% Fetal Bovine Serum (FBS) for 24 hours and reporter assays were performed using the Dual Luciferase reporter assay system (Promega) according to the manufacturer’s instructions. Firefly luciferase activity was normalized to renilla luciferase activity and demonstrated as Wnt reporter activity.NV-Ins Top Luc (Top flash) and NV-Ins Fop Luc (Fop flash) constructs, and renilla supernatant, were generously provided by Dr. Stuart Aaronson lab in Mount Sinai. To generate lentiviral supernatant, 293T cells were co-transfected with 5μg of TOP flash construct with packaging plasmids  when cells reached 80% confluency. Supernatant was harvested and used for transduction of lentiviral particles into MDA-MB-231 and D3H2LN cells, respectively. For transduction, 2X10® Green Supermix (Bio-rad). Results are represented after values were normalized to housekeeping genes (RPL30 or GAPDH) and are presented as fold-differences over control using the ∆∆Ct method for relative quantifications. Each comparison was made using triplicate reactions and in at least 3 experiments. Primer sequences are described in details in Table RNA was prepared using RNeasy Plus Mini kit (Qiagen). For real-time qPCR, 1μg RNA was reverse-transcribed with iScript cDNA Synthesis kit (Bio-Rad). 50-250 ng of resulting cDNA were amplified and analyzed in real time with iTaq™ Universal SYBRCells were plated 40-50 percent confluent in 10 cm plates on the day of transfection. Silencer select non-targeting negative control siRNA (Ambion) or a pool of 4 different siRNAs targeting TGIF1 (40nM)  were used for transfection. Cells were used for invasion assays and qPCR analysis at 48 hour post-transfection.Affymatrix gene microarray was performed in MDA-MB-231 cells that were either untreated or treated with 2.5μM SCR or SID peptide for 24 hours as previously described . Total R-4, SCR versus SID = FDR<1x10-8. We determined Wnt and EMT target genes with significant changes in the H3K4Me3 occupancy by intersecting significantly changed peaks of H3K4Me3 ChIP to ±3 Kb TSS of all Refseq genes, respectively, using Bedtools. Histone modification snapshots were generated using UCSC Genome Browser.The native ChIP-seq data for H3K4Me3 that had been previously published  were useStatistical analyses were performed using a one-way analysis of variance (ANOVA) to compare all pairs of experimental groups. Data were represented as mean ± SEM (or SD) for replicate values. P <0.05 was regarded as statistically significant. For comparison of two groups, two-tailed student’s t tests were used. All the statistical analyses were performed using Graphpad Prism software.


A hallmark of aberrant activation of the Wnt/β-catenin signaling pathway has been observed in most colorectal cancers (CRC), but little is known about the role of non-coding RNAs regulated by this pathway. Here, we found that miR-150 was the most significantly upregulated microRNA responsive to elevated of Wnt/β-catenin signaling activity in both HCT116 and HEK293T cells. Mechanistically, the β-catenin/LEF1 complex binds to the conserved TCF/LEF1-binding element in the miR-150 promoter and thereby transactivates its expression. Enforced expression of miR-150 in HCT116 cell line transformed cells into a spindle shape with higher migration and invasion activity. miR-150 markedly suppressed the CREB signaling pathway by targeting its core transcription factors CREB1 and EP300. Knockdown of CREB1 or EP300 and knockout of CREB1 by CRISPR/Cas9 phenocopied the epithelial-mesenchymal transition (EMT) observed in HCT116 cells in response to miR-150 overexpression. In summary, our data indicate that miR-150 is a novel Wnt effector that may significantly enhance EMT of CRC cells by targeting the CREB signaling pathway. The Wnt/β-catenin signaling pathway plays a central role in normal embryogenesis and development as well as in various human diseases. –3 DysregSince the discovery of the proto-oncogene MYC as a direct transcriptional target of Wnt/β-catenin signaling in 1998, more than one hundred protein-coding genes have been determined to be direct targets of the Wnt pathway.  In additin vitro and in vivo. These results set miR-150 as an effector of Wnt/β-catenin signaling in EMT and the malignant progression of CRC.In this study, we investigated the transcriptional regulation and function of miR-150-5p (referred to as miR-150) that was the most significantly responsive to the activation of Wnt/β-catenin signaling pathway in the LiCl-treated CRC cells. We showed that miR-150 was directly transactivated by β-catenin/LEF1, and overexpression of miR-150 significantly promoted CRC cell migration To identify miRNAs that are responsive to the Wnt signaling pathway, we initiated the study by treating HCT116 and HEK293T cells with LiCl to activate the Wnt/β-catenin signaling pathway. LiCl is commonly used to mimic the activity of Wnt/β-catenin signaling via inhibiting GSK-3β mediated β-catenin phosphorylation. , 20 HCT1A miRNA qRT-PCR array capable of monitoring the expression of 212 highly conserved (between human and mouse) miRNAs was utilized to investigate the miRNA expression profile in LiCl-treated HCT116 and HEK293T cells . This sumut) in HEK293T cells or of LEF1 in HCT116 cells significantly increased miR-150 expression  and mature miR-150. Overexpression of mutant-activated β-catenin  and transcriptional termination site (TTS) for miR-150 were unknown, we first identified the 5′ and 3′ ends of the miR-150 primary transcript by Rapid Amplification of cDNA Ends (RACE). As shown in Figure To determine whether the 5′ flanking region of miR-150 contained a functional promoter, we performed a unidirectional deletion analysis on the approximately 3.4 kb DNA fragment. All the deletion derivatives were cloned into the pGL4.11-basic vector, and luciferase activity was measured in SW480 cells, which harbor a highly activated Wnt/β-catenin signaling pathway. As shown in Figure Studies have proven that the β-catenin/TCF/LEF1 complex regulates the transcriptional activity of target genes by binding to the consensus core TCF/LEF1-binding site (5′-A/TA/TCAAAG-3′) within promoter regions. , 23 To pin vitro, electrophoretic mobility-shift assays (EMSA) were performed by using SW480 cells, which express high endogenous LEF1 and β-catenin  Figure . To valiin vivo, we established a HCT116 cell line stably overexpressing miR-150  or mutant (Mut) putative binding motifs for miR-150 and inserted them downstream of the Renilla luciferase gene in the psiCHECK-2 vector . The 3′UTo determine whether the repression of CREB1 and EP300 accounted for the miR-150-mediated downregulation of the CREB pathway, we analyzed the effects of EP300 and CREB1 knockdown on the CREB pathway. As expected, HCT116 cells transfected with siRNA against EP300 or CREB1 exhibited decreased CREB pathway activity, similar to the effect of miR-150 overexpression Figure . MoreoveImportantly, we observed that activating Wnt pathway by LiCl treatment or LEF1 overexpression in HCT116 cells caused the decrease of CREB1 and EP300 expression, while knockdown β-catenin in SW480 cells had opposite effects, indicating that Wnt pathway could suppress the expression of these two targets Figure  and 4I. The above results prompted us to determine whether the downregulation of CREB signaling mediated the effects of miR-150 overexpression: EMT and the subsequent increased migration of CRC cells. As expected, knockdown of EP300 or CREB1 by siRNA resulted in a similar mesenchymal-like morphological change in HCT116 cells Figure . ConsistIn the present study, we demonstrated an interesting miRNA effector of Wnt signaling, miR-150, that plays a central role in mediating the crosstalk between the Wnt/β-catenin and CREB signaling pathways and contributes to the EMT of CRC cells Figure . AccordimiR-150 was originally found to be specifically and highly expressed in mature B and T cells, where it plays critical roles in normal hematopoiesis and immunity. , 35 Althet al. reported that disrupting EP300 in HCT116 cells resulted in EMT and migration. [From the 45-pathway reporter array analysis, we found that miR-150 overexpression seriously affects multiple signaling pathways for cell growth or proliferation, and CREB was the most downregulated. Importantly, we found that activation of Wnt/β-catenin pathway in HCT116 cells suppressed CREB signaling pathway core factors EP300 and CREB. These findings revealed an unexpected significance of the CREB pathway in colorectal cancer biology, providing evidence in understanding CREB signaling from a new perspective. The CREB signaling pathway participates in various biological processes,  includingration.  These figration.  may act gration. , 56 and gration.  It will Previously, we reported that Wnt/β-catenin signaling can directly transactivate the miR-372/373 cluster in CRC cells to promote cell proliferation and invasion, in part by targeting the Wnt signaling inhibitor Dickkopf-1 (Dkk1).  RecentlyIn addition to miR-150, a cohort of miRNAs have been upregulated following the activation of Wnt/β-catenin signaling in our cell models  and HEK293T were all purchased from the Shanghai Institute of Cell biology, Chinese Academy of Science. HEK293FT cells were obtained from Invitrogen . Cell lines were maintained using standard media and conditions. Establishment of HCT116 subline that stably expressed miR-150 was performed as previously described.  To stimuAll RNA transfections were performed at a final concentration of 50 nM using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction. Transfection of plasmid DNA was performed using Lipofectamine LTX reagent (Invitrogen) for SW480 and HCT116 cells and Lipofectamine 2000 for other cells.et al. [Total RNA was extracted from cells or tumors with TRIzol reagent (Invitrogen) according to the manufacturer's instruction. qRT-PCR assays were performed as previously described.  U6 and Get al.  The primTotal protein was extracted using TRIzol reagent (Invitrogen) as recommended. Protein precipitation was lysed in the buffer containing 1% DTT, 4% CHAPS, 7 M urea, 2 M thiourea and 2% ampholine. Equal total protein extracts were loaded and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto Protran™ nitrocellulose membranes (Whatman). The following antibodies were used for western blot: β-catenin , p-β-catenin , LEF1 , TCF4 , EP300 , CREB1 , E-cadherin , ZO-1 , vimentin , α-tubulin  or GAPDH . GAPDH or α-tubulin was used as the loading control.Cells cultured on coverslips were fixed with 4% Paraformaldehyde and incubated with antibodies against E-cadherin , ZO-1  or vimentin , then stained with secondary antibodies  and nuclear counter-staining with Hoechst 33342 . Fluorescent images were examined using Zeiss 7 DUO NLO .For Immunohistochemistry (IHC), tissues were fixed in 4% formalin for 1 day and followed by paraffin embedding. 4 μm thick serial sections were used for HE staining or IHC-IF. For IHC-IF, primary antibody was used against GFP  and Goat Anti-Chicken IgY H&L  was used as secondary antibody. Then the fluorescence was detected with Zeiss AxioObserver.Z1 (Carl Zeiss).6) were suspended in 200μl 1×PBS and then injected subcutaneously into the dorsal flank of 5-week-old athymic nude mice . The mice were sacrificed 35 days after injection. The primary tumors and livers of mice were dissected, fixed in 4% formalin, and then embedded in paraffin.All procedures for the experiments involving mice were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals , and according to the ethical principles for experiments on animals. HCT116-miR-150 or HCT116-NC stable cells . p<0.05 was considered to be statistically significant.Quantitative data were presented as the mean ± the standard error of the mean (SEM) from a minimum of three independent experiments. Comparisons between two groups were analyzed using the Student's


Beta-catenin/TCF signaling has been reported to promote the growth and metastasis of pancreatic cancer cells. However, the regulation for the beta-catenin/TCF transcriptional complex remains largely unknown. Here, we have found that YEATS4 is a positive regulator for Beta-catenin/TCF signaling. The expression of YEATS4 was elevated in clinical pancreatic cancer samples and pancreatic cancer mouse model. Up-regulation of YEATS4 promoted the growth, migration and invasion of pancreatic cancer cells, while knocking down the expression of YEATS4 inhibited the growth, migration, invasion and metastasis of pancreatic cancer cells. Moreover, the mechanism study revealed that YEATS4 interacted with beta-catenin and activated beta-catenin/TCF signaling. Furthermore, knocking down the expression of YEATS4 impaired the malignant transformation of normal pancreatic cells (HPDE6C7) by the oncogenic Ras. Taken together, our study demonstrated the oncogenic roles of YEATS4 in the progression of pancreatic cancer by activating beta-catenin/TCF signaling and suggested that YEATS4 might be a promising therapeutic target for pancreatic cancer. Numerous studies have demonstrated the roles of Wnt/beta-catenin signaling in cell proliferation, differentiation and motility . Beta-caAberrant activation of Wnt/beta-catenin signaling has been observed in various cancer types –6. NucleYEATS4 was initially found in the nucleoli . It has In this study, we have examined the expression pattern of YEATS4 in pancreatic cancer, investigated the functions of YEATS4 and elucidated the underlying molecular mechanism.G12D), which was driven by the oncogenic RasG12D. The crossing with Pdx-cre deleted the stop condon upstream of RasG12D coding sequence and induced pancreatic intraepithelial neoplasia (PanIN) 8 months after birth. Clearly, the protein level of YEATS4 was increased in the PanIn tissues  by western blot analysis Figure . The effTo explore the underlying mechanisms for the oncogenic roles of YEATS4 in the pancreatic cancer, we screened the effects of YEATS4 on the activity of several pathways using lusiferase reporter assay. Down-regulation of YEATS4 impaired the activity of Topflash reporter  both at the basal level and upon the stimulation of wnt3a Figure . MoreoveNext, we studied how YEATS4 activated the beta-catenin/TCF signaling. Considering the nuclear localization of YEATS4 in the pancreatic cancer, we first tested the interaction between YEATS4 and beta-catenin/TCF4 transcriptional machinery. As shown in Figure Upon the stimulation of wnt3a, beta-catenin and TCF4 formed a complex to activate the expression of downstream genes. We next examined whether he expression of YEATS4 enhanced the interaction between TCF4 and beta-catenin. Forced expression of YEATS4 enhanced the interaction between TCF4 and beta-catenin Figure . Collectin vivo after administration of luciferin (the substrate of luciferase). The HPAC cells knocking down the expression of YEATS4 (HPAC/si YEATS4) and the control cells (HPAC/si con) were injected into the left ventricle of the nude mice and the metastatic foci were monitored. 50 days after the injection, more metastatic foci were observed in the mice injected with the control cells  and normal human pancreatic cell line (HPDE6C7) were obtained from ATCC . All cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (GIBCO), 100units/mL penicillin and 100μg/mL streptomycin in an incubator with 5% COTotal RNA was exacted from the clinical tissues using TRIzol. The reverse transcription kit was used to prepare the complementary DNA (cDNA). The expression of YEATS4 in the pancreatic cancer tissues and paired normal tissues was examined by quantitative real-time PCR using SYBR® Green Realtime PCR Master Mix (TOYOBO) following the instructions of the manufacturer. Sequences of quantitative real-time PCR primers are listed as follows: 18S Forward primer: 5′-TAAATCAGTTATGGTTCCTT -3′; 18S Reverse primer: 5′-CGACTACCATCGAAAGTTGA-3′; YEATS4 Forward primer: 5′-AGAGAATG GCCGAATTTGGG-3′; YEATS4 Reverse primer: 5′-TCATAGAACTCTGAAACCAC-3′Xylene and ethanol was used to deparaffinized and rehydrated the paraffin-embedded tissue. Endogenous peroxidase activity was blocked with 0.35% H2O2 solution. Antigens retrieve was performed using microwaving. Non-specific binding was blocked by 1% BSA solution Sections were stained with YEATS4 antibody and visualized with secondary antibody . Slides were then developed with DAB andcounterstained with hematoxylin.The cellular proteins were resolved by SDS-PAGE after lysed by the RIPA buffer. The proteins were transferred to the PDVF membrane. After blocking with the 3% BSA solution, the membrane was incubated with following primary antibodies over night: anti-YEATS4 (Abcam), anti-Snail , anti-Cyclin D1 and anti-c-Myc , anti-GAPDH (Santa Cruz). The membranes were washed with TBST solution and incubated with the secondary antibody for 1 hour at the room temperature. The protein was visualized by ECL kit.The coding sequence of beta-catenin was cloned into the expression vector pGEX-4T-1. The fusion protein GST-beta-catenin was purified. The whole cell lysates of HPAC were prepared in 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 0.1% NP40 and protease inhibitor cocktail. 5 μg GST-beta-catenin fusion protein and 500 μg cell lysates were incubated at 4°C over night. 50 μl of glutathione-Sepharose-4B beads were added to the samples and incubated at 4°C for 1 hr to capture the GST fusion proteins. After washing with lysis buffer three times, the proteins were eluted in Laemmli buffer and analyzed by SDS-PAGE.HPAC Cells were grown to a subconfluent density. 16 hours later, the reporter assays were performed using 0.1 μg of Topflash, 0.5 μg of expression vector, and 0.05 μg of TK Renilla luciferase . 48 hours later, cells were treated with Wnt3a protein for 8 hours. Then, cell lysates were prepared and the reporter activity was measured using the dual-luciferase reporter assay system (Promega).The coding sequence of YEATS4 was amplified by PCR and inserted into the expression vector pcDNA3.1 to obtain the myc tagged YEATS4. The coding sequence of beta-catenin was amplified by PCR and inserted into the expression vector pCMVTag2B to obtain the Flag tagged beta-catenin. The coding sequence of TCF4 was amplified by PCR and inserted into the expression vector pCMV-HA to obtain the HA tagged TCF4.RNAi lenti-virus particles (sicon and si YEATS4) were purchased from GeneChem (China). Cells were infected with the indicated lenti-virus particles of the same MOI for 24 hours and then stable knock-down cells were selected with the medium containing puromycin for at least a week.5 cells/well were plated in 96-well plates. Cell growth was determined using the 3--2,5-diphenyltetrazolium bromide (MTT) colorimetric growth assay for a week. Every other day, cell growth was determined by adding MTT solution (50μg/well) for 4h. Cellular MTT was resolved with DMSO and was measured at 540 nm. All experiments were performed in triplicates.105) suspended in 0.05ml medium containing 1% FBS were placed in the upper chamber, and the lower chamber was loaded with 0.152ml medium containing 10% FBS acting as the chemoattractant. 12 hours later, cells migrated to the lower surface of filters was detected with traditional hematoxylin and eosin (H&E) staining. The experiments were repeated for three times. Five random visual fields were counted for each sample and the average was determined.Boyden chamber was used to evaluated the motility of pancreatic cells. Cells  in 6-well plates. The plates were coated with bottom layer (0.5% agarose and 10% FBS in DMEM). After 14 days of incubation, the colonies were counted and measured. All of the experiments were done at least three times.G12DKras and Pdx-Cre mice were obtained from Jackson Lab . Pancreatic cancer mouse models G12DPdx-Cre; Kras mice were generated by crossing G12DKras and Pdx-Cre mice.Mice were housed and treated after being approved by the Institutional Animal Care and Use Committee of Jiangsu University. t-test (two-tailed) using Prism GraphPad software. Differences with P < 0.05 were considered statistically significant. Data were represented as mean±SEM.Statistical analysis was performed by the Student


Methoxetamine (MXE) is a ketamine analogue with a high affinity for the N-methyl-D-aspartate (NMDA) receptor. MXE is a newly emerging designer drug of abuse and is widely available through on-line sources and is not detected by routine urine drug screens. In this report, we describe a United States (US) veteran with posttraumatic stress disorder (PTSD) and heavy polysubstance use, who injected high dose MXE for its calming effect. Given MXE's structural similarities to ketamine and recent work showing that ketamine reduces PTSD symptoms, we hypothesize that MXE alleviated this veteran's PTSD symptoms through action at the NMDA receptor and via influences on brain-derived neurotrophic factor (BDNF). To our knowledge, this is the first case report of self-reported use of MXE in the US veteran population. More awareness of designer drugs, such as MXE, is an important first step in engaging patients in the treatment of designer drug addiction in both military/veteran settings and civilian settings. We present a case of a young male United States (US) combat veteran with posttraumatic stress disorder (PTSD) and a complex substance use history, which included opioid use disorder treated with methadone, stimulant use disorder, and stimulant-induced psychosis. Initially unknown to clinical providers, he was also injecting large quantities of the designer drug methoxetamine (MXE) for its perceived calming effect. To our knowledge, this is the first case report of self-reported use of MXE in a US veteran .Mr. A is a 29-year-old US veteran with chronic PTSD (100% service-connected), heavy polysubstance use , cannabis use disorder, and tobacco use disorder), as well as unspecified depressive and anxiety disorders, childhood history of attention deficit/hyperactivity disorder, childhood sexual abuse, HIV on antiretroviral therapy, chronic hepatitis C, and tinnitus. He received care through the Veterans Affairs Medical Center (VAMC) and became a patient in the Opioid Treatment Program (OTP).Three years after enrolling in the OTP, Mr. A began to describe symptoms of psychosis. These symptoms included hearing voices which talked about him, speaking his thoughts, and feeling as though someone was following him. His urine toxicology screen was positive for amphetamines, cocaine, cannabis, and opiates. Psychosis was treated with risperidone. Other medications included methadone 140 mg daily, emtricitabine 200 mg/tenofovir 300 mg daily, and raltegravir 800 mg daily. Over the ensuing six months, he was admitted to the VAMC psychiatric unit five times for treatment of acute psychosis. Medication trials for psychosis, depression, and anxiety included olanzapine, aripiprazole, mirtazapine, lorazepam, and gabapentin. Mr. A's methadone dose varied throughout this period depending on his concurrent substance use. At one point, he self-tapered methadone to discontinuation, although he later restarted it.It was hypothesized that Mr. A's heavy use of stimulants precipitated the development of psychosis. This was based on the substances that he had reported using, the substances that were present on urine drug screens, and the observation that psychosis diminished rapidly during each hospitalization. Intermittent erythema and edema of his feet were noted, which were consistent with cellulitis and treated with antibiotics. Neurological examinations were normal during these episodes of cellulitis. Puncture marks were present on the dorsum of his left hand. There were no other abnormalities noted on physical examination.Four years after enrolling in the OTP, Mr. A disclosed he was injecting MXE obtained from an on-line source. He described using MXE intermittently for the past year and had progressed to using 50–70 mg daily. He had tried ingesting it, but he preferred either intravenous or intranasal routes. When using MXE, he described feeling as if he was “being with a Buddha.” He preferred MXE to illicit use of ketamine, as he found MXE allowed him to feel more “spiritual” and “calm.” He believed that MXE relieved the distress and dysphoria of withdrawal that accompanied his methadone tapers, and he felt that using MXE allowed him to taper methadone more rapidly. The VAMC toxicology laboratory lacked the capability to detect MXE in urine. Thus, while urine drug screens returned positive for other substances, MXE was not identified.Now five years after enrolling in the OTP, Mr. A continues to use MXE  along with other substances . Opioid replacement therapy was restarted, and his current dose of methadone is 50 mg daily. He is also prescribed olanzapine 15 mg twice daily to treat symptoms of psychosis.The discussion focuses on this veteran's PTSD and self-reported use of MXE to produce spiritual experiences and feelings of calm. This focus was chosen because, despite Mr. A's concurrent use of other substances with the potential to calm or produce spiritual effects, Mr. A attributed these effects to MXE. These effects were not reported prior to the addition of MXE to the plethora of substances he reported using. Mr. A's use of what he believed to be MXE was based on self-report. Novel psychoactive substances often contain compounds other than or in addition to those advertised , 3.Mr. A is one of more than 1.8 million US troops who served in Operation Enduring Freedom (OEF) and/or Operation Iraqi Freedom (OIF) since combat began in 2001. Large-scale studies have found that 10–18% of OEF/OIF veterans are likely to have PTSD following deployment . SubstanCommonalities exist between neurotransmitters, brain regions, and neurocircuitry involved in PTSD and comorbid SUD . SeveralOther neurochemicals interact with the HPA axis in both PTSD and SUDs . Brain-dBDNF signaling impacts the NMDA receptor, an ionotropic receptor located throughout the brain and involved in learning, memory, and long-term potentiation . BDNF inMXE is a ketamine analogue . MXE is Not only did Mr. A suffer from chronic PTSD, he also suffered from the perceptual and cognitive impact of psychosis and the psychological and physical effects of sustained, heavy polysubstance use. Searching for a way to quiet this profound internal chaos, we propose Mr. A sought the calming effects of MXE. These effects served multiple purposes: (1) reducing the fear and anxiety associated with PTSD and psychosis; (2) mitigating the anxiety associated with the activation of the sympathetic nervous system due to opioid withdrawal; and (3) mitigating the anxiety and dysphoria due to the chronic activation of the HPA axis.Given that MXE is a ketamine analogue, we suggest the anxiolytic and antidepressant effects resulted in part from an increased translation of BDNF, which in turn increased the expression of NMDA receptors and NMDA receptor activity. Blockade of these receptors by MXE resulted in anxiolysis and reduction of depression and PTSD symptoms , analogoIn summary, this is the first case report of self-reported use of MXE in a US veteran. This veteran found MXE to exert calming effects and to allow spiritual experiences, which we hypothesize were due in part to increases in BNDF, its effect on the NMDA receptor, and MXE-induced antagonism of the NMDA receptor. Regarding military/veteran populations, a previous report commented that MXE may “threaten military readiness” . This ca