Aberrant STAT1 signaling is observed in human hepatocellular carcinoma (HCC) and has been associated with the modulation of cell proliferation and survival. However, the role of STAT1 signaling in HCC and its underlying mechanism remain elusive.We transiently transfected pcDNA3.1-STAT1 and STAT1 siRNA into SMMC7721 and HepG2 cells. Western blot and qRT-PCR examined the expression of protein and RNA of target genes. Cell viability was assessed using MTT assay, and cell cycle and apoptosis were analyzed by flow cytometry.We found that STAT1 overexpression increased protein expression of p53 and Fbxw7, and downregulated the expression of cyclin A, cyclin D1, cyclin E, CDK2, Hes-1 and NF-κB p65. These changes led to growth inhibition and induced G0/G1 cell cycle arrest and apoptosis in SMMC7721 and HepG2 cells. Conversely, ablation of STAT1 had the opposite effect on p53, Fbxw7, Hes-1, NF-κB p65, cyclin A, cyclin D1, cyclin E and CDK2, and improved the viability of SMMC7721 and HepG2 cells.Our data indicate that STAT1 exerts tumor-suppressive effects in hepatocarcinogenesis through induction of G0/G1 cell cycle arrest and apoptosis, and may provide a basis for the design of new therapies for the intervention of HCC in the clinic. Hepatocellular carcinoma (HCC) accounts for 80–90 % of liver cancers and is the eighth most commonly occurring cancer in the world . EpidemiThere is compelling evidence that STAT1 play an important role in promoting apoptotic cell death, and has been supported by the findings that growth inhibiting and pro-apoptotic activities of interferon-gamma (IFN-γ) are largely mediated by STAT1 signaling . It has In this present study, we tested the impact of induction or knockdown of STAT1 expression on the proliferation, apoptosis and cell cycle of HCC cell line SMMC7721 and HepG2. We have demonstrated that STAT1 upregulation can significantly inhibit the in vitro growth of SMMC7721 and HepG2. Conversely, STAT1 knockdown promoted proliferation in the same cell line. STAT1-induced growth suppression is at least partially due to apoptosis and G0/G1 cell cycle arrest. Consistent with cell cycle arrest, expression levels of cyclin A, cyclin D1, cyclin E, and CDK2 protein all decreased. Upregulation of p53 and Fbxw7 expression and downregulation of NF-κB p65 and Hes-1 were observed, and may be related to STAT1-induced apoptosis. Therefore, Our data suggest that STAT1 may be a negative regulator of the development and progression of human HCC growth through induction of apoptosis and cell cycle arrest.SMMC7721 and HepG2 cells were engineered to transiently express high levels of a recombinant plasmid encoding the STAT1 sequence (pcDNA3.1-STAT1) or control empty vector (EV) pcDNA3.1. The levels of STAT1 expression were determined by qRT-PCR and western blot assays. Compared with the control, the levels of STAT1 proteins in the pcDNA3.1-STAT1-transfected cells showed higher expression than that in the EV and unmanipulated cells  belong to a family of seven cytoplasmic proteins that function as signal messengers and transcription factors participating in cellular responses to cytokines and growth factors , 17. AmoIn this study, we transiently transfected STAT1 expression vectors and STAT1-specific siRNA into SMMC7721 and HepG2 cells to investigate the function of STAT1 in driving HCC development or progression. We observed that STAT1 overexpression caused growth inhibition, induced G0/G1 cell cycle arrest and promoted apoptosis in SMMC7721 and HepG2 cells, whereas ablation of STAT1 improved viability. The role of the STAT1 cascade in tumor is controversial, and despite strong data indicating that STAT1 downregulation was most prominent in the tumor cells themselves when compared with the surrounding stroma and infiltrating lymphocytes , 20. StuApoptosis and cell cycle arrest involve complex molecular cascades, and dysfunction of various genes may lead to the onset and progression of apoptosis and cell cycle arrest. We explored the mechanism of STAT1-induced apoptosis and cell cycle arrest by assessing the effect of STAT1 on the expression of apoptosis-related factors  and cell cycle regulators  in SMMC7721 and HepG2 cells. It is well recognized that STAT1 promotes cell death through mechanisms that are dependent upon transcriptional activation of genes encoding proteins involved in modulating or promoting cell death, such as caspases, death receptors and ligands, iNOS, and Bcl-xL, as well as those involved in cell cycle arrest, such as p21 WAF1. It is also interesting to note that STAT1 can associate with proteins that are directly or indirectly involved in apoptotic cell death, including TRADD, p53, HATs and HDAC. Previous studies have demonstrated that overexpression of mutant or wild-type p53 can result in apoptotic cell death and that p53 can cause cell cycle arrest by transcriptionally upregulating p21 . Fbxw7 iIn summary, our data indicated that STAT1 may function as a suppressor of HCC cell proliferation, and a regulator of HCC cell apoptosis and cell cycle arrest. Hence, our findings may provide a basis for the design of new therapies for the intervention of HCC in the clinic.2. The plasmids of pcDNA3.1 (EV) and pcDNA3.1-STAT1 were obtained from GenePharma. pcDNA3.1 (EV) and pcDNA3.1-STAT1 were transfected into SMMC7721 and HepG2 cells at 70 % confluence using X-tremeGENE HP DNA transfection reagent  according to the manufacturer’s protocol. Cells were transfected for 48 h in 6-well plates and then analyzed for flow cytometry, MTT assay, western blot and qRT-PCR.SMMC7721 and HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal calf serum at 37 °C and 5 % CO5/well) were cultured in 6-well plates at 70 % confluence, and transfected in duplicate with 1.3 µg each type of STAT1-specific or control siRNA using X-tremeGENE siRNA transfection reagent  according to the manufacturer’s instructions. Cells were transfected for 48 h in 6-well plates and then analyzed for flow cytometry, MTT assay, western blot and qRT-PCR.The sense and antisense sequences of the STAT1-specifc and control siRNAs were synthesized by GenePharma, Shanghai, China and are present in Table After transfection with the corresponding STAT1 vectors or siRNAs for 48 h, the cells were harvested and washed twice with cold PBS, then fixed in 75 % alcohol for 2 h at 4 °C. After washing in cold PBS three times, cells were resuspended in 1 mL of PBS solution with 40 μg of propidium iodide (Sigma) and 100 μg of RNase A (Sigma) for 30 min at 37 °C and analyzed with the BD Accuri C6 system . The distribution of cells in different phases of the cell cycle was calculated using the Modifit LT software.After transfection for 48 h, the cells were harvested and washed three times with cold PBS. Cells were then resuspended in the staining buffer and stained using the Annexin V-FITC Apoptosis Assay Kit  according to the manufacturer’s instructions. The stained cells were analyzed by flow cytometry . Annexin V-positive and propidium iodide-negative cells were counted as apoptotic cells.4 cells per well in 100 μL complete medium. After transfection with the corresponding plasmid vector or siRNA, 10 μL MTT solution (5 mg/mL) was added, and cells were incubated for an additional 4 h. Subsequently, 100 µL of MTT solubilization buffer was added to the wells. Following a 10-min mixing period, the absorbance was analyzed at 570 nm using a microplate reader. The background absorbance at 690 nm was subtracted from the 570 nm measurement. Each experiment was performed in triplicate, and the mean value was calculated.Cell viability was assessed using the MTT colorimetric assay . SMMC7721 and HepG2 cells were seeded in 96-well plates at a density of 1 × 10® First Strand cDNA Synthesis Kit  and random oligodeoxynucleotide primers. PCR amplification was performed in 20 μL reactions containing cDNA generated from 2 ng of the original RNA template, 400 nmol/L of each gene-specific forward and reverse primer, 10 μL of 2 × SYBR® Premix Ex Taq™ II , 0.4 μL of ROX reference dye, and 6.0 μL of dH2O. Amplified signals were detected using the ABI PRISM 7300 Real-Time PCR system . The annealing temperature was optimized using the temperature gradient program that is part of the ABI PRISM software. Experiments were performed in duplicate. The relative target mRNA levels were determined using the 2ΔΔCt method. The primer sequences used are summarized in Table Total RNA was isolated from cultured cells using Trizol  according to the manufacturer’s instructions. RNA concentration and purity were determined from absorbance measured at both 260 and 280 nm. RNA 1 μg) was reverse transcribed using the PrimeScript μg was rProtein concentrations were determined using the BCA Protein Assay  according to the manufacturer’s instructions. Samples containing equal amounts of protein (40 μg) were resolved by 10 % SDS-PAGE and transferred to nitrocellulose membranes. After blocking for 2 h at room temperature with TBS-T  containing 5 % skim milk, and probed at 4 °C overnight with rabbit anti-STAT1 , rabbit anti-p53 (1:400), rabbit anti-Fbxw7 (1:400), rabbit anti-cyclin A (1:400), rabbit anti-cyclin D1 (1:400), rabbit anti-cyclin E (1:400), rabbit anti-CDK2 (1:400) , rabbit anti-NF-κB p65 (1:2000), rabbit anti-Hes-1 (1:2000) , and mouse anti-actin (1:8000). Proteins were detected by exposing the blots to X-ray film (Kodak).2 test was used to analyze the flow cytometry data. P values less than 0.05 were considered statistically significant.SPSS version 17.0 software was used for all statistical analyses. All of the results are expressed as the mean ± SD. Statistical analysis was performed using standard two-way ANOVA for repeated measurements, and the Χ


The gene expression of CHOP and Ero1-α was measured using Quantitative-PCR (Q-PCR) in rat hippocampi following global cerebral ischemia, and in hypoxic pheochromocytoma cells during normothermic (37 °C) and hypothermic (31 °C) conditions. As a result of ischemia, a significant increase in expression of CHOP and Ero1-α was observed after three, six and twelve hours of reperfusion following global ischemia. A stable increase in CHOP expression was observed throughout the time course , whereas Ero1-α expression peaked at three to six hours (p < 0.0001). Induced hypothermia in hypoxia stressed PC12 cells resulted in a decreased expression of CHOP after three, six and twelve hours (p < 0.0001). On the contrary, the gene expression of Ero1-α increased as a result of hypothermia and peaked at twelve hours (p < 0.0001). Hypothermia attenuated the expression of CHOP, supporting that hypothermia suppress endoplasmic reticulum stress induced apoptosis in stroke. As hypothermia further induced up-regulation of Ero1-α, and since CHOP and Ero1-α showed differential regulation as a consequence of both disease (hypoxia) and treatment (hypothermia), we conclude that they are regulated independently.Hypoxia induced endoplasmic reticulum stress causes accumulation of unfolded proteins in the endoplasmic reticulum and activates the unfolded protein response, resulting in apoptosis through CCAAT-enhancer-binding protein homologous protein (CHOP) activation. In an  Cischemia ,6,7,8,9.Cerebral ischemia causes hypoxic stress in cells, leading to endoplasmic reticulum (ER), environment disruption, and accumulation of unfolded proteins in the ER lumen. In response to cellular aggregation of misfolded and/or unfolded proteins, the Unfolded Protein Response (UPR) becomes activated ,11. The In unstressed cells, the ER chaperone glucose-regulated-protein (GRP78) binds pancreatic-ER-kinase (PERK), inositol-requiring ER-transmembrane RNAse-1 (IRE-1) and activating-transcription factor-6 (ATF6), keeping them inactivated . Upon stCHOP plays a central role in the ischemic damage resulting in neuronal death . Many CH2+ release, initiating apoptosis [Endoplasmic reticulum oxidoreductin-α (Ero1-α) has also been suggested to be a CHOP target gene . Ero1 expoptosis . Thus, tin vitro model, we mimicked the ischemic condition by introducing hypoxia into a neuron-like cell line and applied hypothermic conditions to evaluate the effects of hypothermia on the transcriptional levels of these mediators.In an experimental animal model of global ischemia, we investigated relative changes in the expression of CHOP and Ero1-α following ischemia. Subsequently, in an Sixty male Wistar rats  obtained from Taconic  were cared for according to the guidelines and approvals of the Department of Experimental Medicine, University of Copenhagen, Denmark, and all the experiments were conducted according to the Danish Animal Experiments Committee (#2012-DY-2934). Animals were acclimatized for 7 days prior to surgery and allowed free access to food and water under diurnal lighting conditions. All efforts were made to diminish pain, suffering and stress of the animals.2/21% O2). Cells were plated at a density of 1–2 × 104 cells/cm2.PC12 cells are a commercially available rat pheocromocytoma cell line originating from rat adrenal medulla . PC12 cells were grown on collagen  coated (6 ug/mL) dishes with DMEM , supplemented with horse serum (Life technologies), fetal bovine serum (Life technologies) and penicillin/streptomycin (Life technologies) (10/5/1%) and cultured in a standard incubation chamber . As global brain ischemia may impede respiratory system functions, ventilator-controlled respiration was performed with a Harvard 683 small animal ventilator . Both common carotid arteries were gently exposed and systemic hypotension (50 mmHg) was induced with a heparinized syringe through the femoral catheter. Arteries were then fully ligated for 12 min. with subsequent removal of ligatures and reperfusion of 3, 6 and 12 h (n = 10/group). Incision wounds were treated with lidocain gel . At the end of reperfusion, rats were deeply anesthetized, decapitated, and brains were rapidly removed, snap frozen in isopentane and stored at −80 °C. Dorsal and ventral parts of the hippocampus were carefully dissected on ice and added TRIzol reagent (Invitrogen) to avoid RNA degradation prior to RNA isolation.Rats were subjected to experimental global ischemia by the two-vessel occlusion (2-VO) model during systemic hypotension . Anesthe−5 M thapsigargin (Sigma-Aldrich) for 1 h, 4 h or 24 h at either 37 °C or 31 °C in a standard incubator, in order to establish a positive control for UPR activation in the cell line. To identify suitable working concentrations, a survival MTT  assay was performed with 10 different thapsigargin concentrations (0.02 × 10−5 M to 10.24 × 10−5 M). A control sample of untreated PC12 cells with the lowest corresponding DMSO concentration (0.031%) was incubated for 24 h at 37 °C. One biological sample for each treatment parameter was generated.Confluent PC12 cells were incubated with 0.32 × 102, controlled with nitrogen.Three biological samples of PC12 cells (grown to confluency at 37 °C) were incubated at 37 °C or 31 °C for 1 h, 3 h, 6 h or 12 h in a hypoxic incubation chamber with 0.3% O2) at 37 °C. Biological triplicates were generated for each treatment parameter (time/temperature). At the end of incubation time, cells quickly proceeded to RNA isolation.Three biological control samples were incubated for 12 h in a standard incubator (5% CORNA extraction was performed according to the TRIzol reagent protocol (Life Technologies) and measured on a BioPhotometer 6131 spectrophotometer . RNA samples were reverse transcribed using the ImProm-II Reverse Transcription System . Thermal cycling conditions were set to 5 min at 25 °C for annealing, 60 min at 42 °C for extension and 15 min at 70 °C for inactivating the reverse transcriptase enzyme. From this reaction, a cDNA concentration of 0.05 μg/μL was obtained.All samples were prepared in triplicates for the targets CHOP, Ero1-α, and Hmbs . Primers for the housekeeping gene Hmbs, HPRT, synaptophysin, and β-actin were tested for stability during hypoxia under normo- and hypothermic conditions. Hmbs exhibited highly stable expression, which is why this gene was chosen as the normalization target for further studies.2O, 7.5 μL relevant primers , and 12.5 μL RealQ-PCR dUTP Master Mixes Kit  was used for a total of 25 μL. Thermal cycling conditions were: fifteen minutes at 95 °C (for hot start of the Taq DNA polymerase enzyme), forty cycles at 95 °C for 30 s (dissociation of cDNA strings), 60 °C for 1 min , and 72 °C for 30 s (extension). The Q-PCR reactions were performed using the ABI 7300 Detection System (Life Technologies). In a 25 μL reaction, 2 μL cDNA (100 ng/2 μL), 3 μL nuclease free HDissociation curves were included in all runs—one cycle of 95 °C for 30 s, 50 °C for 30 s, and from 50 °C to 95 °C, with a reading every 0.5 °C and a hold for 10 s.Primers used: Hmbs (76 base pairs (bp), F: TCTAGATGGCTCAGATAGCATGCA, R: TGGACCATCTTCTTGCTGAACA), CHOP  and Ero1-α .p < 0.05 was considered statistically significant.All data are calculated according to the comparative Ct method and expressed as mean ± SD, presenting data as fold change in gene expression normalized to housekeeping gene (Hmbs) and relative to the untreated control, an approach determining the relative quantification rather than absolute quantification . Ordinarp < 0.01). At 6 h of reperfusion, CHOP levels were further increased (p < 0.0001), however not significantly different from the 3 and 12 h group. Similar to CHOP, Ero1-α was also increased at 3 to 6 h following ischemia (p < 0.0001), although it seemed to reach maximum at this time point and was reduced towards normal levels after 12 h of reperfusion. Nevertheless, 3, 6 and 12 h after ischemic injury all show significant up-regulation of Ero1-α compared to untreated controls (p < 0.0001 and 0.01 respectively).Induction of global ischemia altered the expression level of CHOP and Ero1-α in hippocampi of Wistar rats . A stablThapsigargin were used in order to verify the suitability of PC12 cells in our experimental setup. Both CHOP a and ERO2+-ATPase) pump inhibitor, thapsigargin, a suitable concentration for induction of a UPR response in the PC12 cell line was found (data not shown), confirming the ability of the cell line to activate UPR upon stress. Since we saw a substantial increase in both CHOP and Ero1-α expression levels at 3 h in our in vivo model, we included an additional 1 h group subjected to hypothermic conditions. Messenger RNA expression of the two UPR markers was unaffected after 1 h of hypoxia, whereas 3, 6, and 12 h of hypoxia showed similar expression patterns as seen in vivo . In contrast, expression levels of Ero1-α were increased as a result of hypothermia at 6 h (p < 0.001) and 12 h (p < 0.0001) (With the use of a SERCA (sarco/endoplasmatic reticulum Ca in vivo . Howevervo model . Ero1-α  0.0001) .In our study, global cerebral ischemia was associated with increased transcriptional levels of CHOP and Ero1-α. Whereas high CHOP expression was maintained throughout the study period, ERo1-α peaked at 3 to 6 h (ischemia) and 6 h (hypoxia), respectively. Additionally, we found that hypothermia (31 °C) during hypoxia modulated the ER stress response through downregulation of CHOP after 3, 6 and 12 h and up-regulation of Ero1-α after 6 and 12 h. The fact that these two genes are differentially expressed, both as a consequence of disease induction (hypoxia) and treatment (hypothermia), suggests that they are independently regulated.in vivo [in vitro [Current research suggests that hypothermia causes alterations in the expression of ER stress related genes. Amongst these, an increased expression of the protective GRP78 has been identified, and decreased expression of proapoptotic factor CHOP and, thereby, a reduction in apoptosis ,20. Furtin vivo ,20 and iin vitro . Ero1-α in vitro ,22,23.in vivo [in vitro results. An increase in CHOP expression up to 24 h following global ischemia, associated with death of hippocampal neurons, has previously been shown . Furtherin vivo  in agreeIn contrast, an augmentation of Ero1-α in hypoxic cells at hypothermic conditions was observed. To our knowledge, investigations of the role of Ero1-α in apoptotic pathways following ischemia as well as the influence of hypothermia are lacking. Ero1-α is assumed to be regulated through hypoxia-inducible-factor-1 (HIF-1), a cytosolic transcription factor which is increased in response to low oxygen tension, while Ero1-β is assumed to be regulated through the UPR ,23. Furtin vivo. Another shortcoming of the study is that our results only address the transcriptional levels. In a broader translational perspective, experiments with the use of transgenic mice relating ischemic damage to specific UPR proteins may highlight whether regulating proteins of the UPR truly is neuroprotective.It should be noted that the PC12 cell line is derived from electrically inexcitable pheochromocytes. Therefore, the results seen in our PC12 cells, in particular the lack of downstream CHOP effects on Ero1-α, should be further confirmed in neurons in vivo and in vitro model of ischemic stroke, we provide evidence that CHOP and Ero1-α of the UPR are possible therapeutic targets in ischemic injury. Hypothermia seems to result in a down-regulation of the pro-apoptotic factor CHOP and an increase in Ero1-α expression, thus indicating that hypothermia decouples CHOP-induced transcription of Ero1-α. Our results draw attention to the potential effect of hypothermia on the UPR and future studies of CHOP and Ero1-α regulation at the protein level will further substantiate their feasibility as drug targets in stroke treatment.In agreement with other studies, we show that an ischemic insult results in significant changes in the transcriptional levels of ER stress regulators ,24. Our


Purpose. To investigate the effect of 2-Methoxyestradiol (2ME2) on experimental autoimmune uveitis (EAU) and the mechanism. Method. C57BL/6 male mice were used to establish the EAU model. 2ME2 was abdominal administrated in D0–D13, D0–D6, and D7–D13 and control group was given vehicle from D0–D13. At D14, pathological severity was scored. Lymphocyte reaction was measured using MTT assay. T cell differentiation in draining lymph nodes and eye-infiltrating cells was tested by flow cytometry. Proinflammatory cytokines production from lymphocytes was determined by ELISA. Result. The disease scores from 2ME2 D0–D13, 2ME2 D0–D6, 2ME2 D7–D13, and vehicle groups were 0.20 ± 0.12, 1.42 ± 0.24, 2.25 ± 0.32, and 2.42 ± 0.24. Cells from all 2ME2 treated groups responded weaker than control (p < 0.05). The inhibitory effect of 2ME2 on lymphocyte proliferation attenuated from 2ME2 D0–D13 to 2ME2 D0–D6 and to 2ME2 D7–D13 groups (p < 0.05). 2ME2 treated mice developed fewer Th1 and Th17 cells both in draining lymph nodes and in eyes than control (p < 0.05). Lymphocytes from 2ME2 group secreted less IFN-γ and IL-17A than those from control (p < 0.05). Conclusion. 2ME2 ameliorated EAU progression and presented a better effect at priming phase. The possible mechanism could be the inhibitory impact on IRBP specific lymphocyte proliferation and Th1 and Th17 cell differentiation. Uveitis is a group of diseases among the leading causes of visual deficit and blindness. Uveal and retinal layers as well as vitreous cavity could all be affected within disease progression. Clinically, this group of diseases can be divided into infectious and noninfectious causes, the latter one is known as autoimmune uveitis . Due to γ) can activate macrophages, while Th17 cells by secreting interleukin 17A (IL-17A) promote inflammatory responses [Different immune cells subsets play important roles in disease development of EAU. Th1 and Th17 responses and relative cytokines are believed to be pathogenic in tissue damage , 5. Th1 esponses , 7. Manyesponses .Endogenous metabolite of estrogen, 2-Methoxyestradiol (2ME2), is a promising antiangiogenic and antitumor compound –12. BesiIn this study, we demonstrated that the endogenous methylated estrogen, 2ME2, could taper autoimmune uveitis development in a time-dependent manner. The disease modifying effect was established on reducing antigen specific T cell proliferation, inhibiting T cell differentiation and related proinflammatory cytokines secretion.6~8-week-old C57BL/6 mice were purchased from Shanghai Laboratory Animal Center, CAS (SLACCAS), and housed in a specific-pathogen-free condition with water and standard laboratory chow ad libitum. Animal care and use were in compliance with the Institutional Animal Care and Use Committee Guidelines and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Mycobacterium tuberculosis (Difco). Three peptides were used: 1–20 300 μg, 461–480 200 μg, and 651–670 200 μg per mouse. 2ME2 purchased from MedChem Express was dissolved into DMSO (Sigma). Roswell Park Memorial Institute 1640 (RPMI1640) medium was purchased from Gibco. MTT kit was purchased from Sigma. ELISA kit was purchased from eBioscience. CD3, CD4, IL-17A, and IFN-γ antibodies used in flow cytometry were purchased from BD. Other reagents and materials used in cell culture, flow cytometry, and HE stain were purchased from Sigma unless otherwise specified.Three peptides of IRBP protein were employed to formulate the antigen complex. They were 1–20 (SGIPYIISYLHPGNTILHVD), 461–480 (LRHNPGGPSSAVPLLLSYFQ), and 651–670 (LAQGAYRTAVDLESLASQL) which C57BL/6 mice were sensitive to . PeptideC57BL/6 mice were immunized subcutaneously 0.1 mL at tail and 0.05 mL at both thigh sites with IRBP antigen complex. 500 ng Pertussis toxin was injected concurrently. This day was settled as day 0. Then mice were divided into 4 groups, each group containing 5 mice. 15 mg/kg 2ME2 or vehicle was abdominal injected during 0–13 days, 0–6 days, and 7–13 days. At day 14 eyes or lymphoglandula was collected after euthanasia.μm sections were stained with standard H&E. Eye sections cut through pupillary-optic nerve planes were scored in a masked fashion. Severity of EAU was graded on a scale of 0–4 in half-point increments using the criteria described previously [Eyes harvested 14 days after immunization were prefixed in 4% phosphate-buffered glutaraldehyde for 1 h  and then transferred to 10% phosphate-buffered formaldehyde until processing. Fixed and dehydrated tissue was embedded in methacrylate, and 4 to 6 eviously , based oγ, and IL-17A [™ (BD).Eyes' external tissue was trimmed after enucleation; incision was made along the limbus to remove the lens. The rest of the eye was minced into small pieces and digested in 1 mg/mL collagenase IV in RPMI containing 10% FCS for 1 hour at 37°C. After trituration and filtration, the resultant cell pellet was resuspended in RPMI with 10% FCS and seeded in 48-well plate. Harvested lymph nodes were dispersed by trituration in RPMI with 10% FCS on ice, followed by filtration and washing. Then single-cell suspension was obtained and seeded in 96-well plate for intracellular cytokine evaluation and proliferation assay. PMA (50 ng/mL) and ionomycin (500 ng/mL) together with brefeldin A were added to pulse both the eye-infiltrating cells and lymphocytes for 5 hours. Cells were then fixed with 2% paraformaldehyde, permeabilized in 0.5% saponin, and stained with CD4, IFN-d IL-17A . Analysi∗106/well and maintained with 200 μL culture media and 5 μg/mL IRBP for 48 hours in 96-well plates. 80 μL MTT was then added to each well and cells were further incubated for 3 hours at 37°C. Supernatants were then removed and 100 μL of chromogenic reagent was added to each well. After shaking in darkness for 10 min, a spectrophotometer was used to measure absorbance at 540 nm. Three independent experiments were performed.MTT assay was used to determine the effect of IRBP on cell proliferation. Cells from 4 groups previously described were seeded 1.5γ concentration according to the manuals from the manufacturer. Briefly, ELISA plate was coated with 100 μL/well of capture antibody in 1x coating buffer, sealed, and incubated overnight at 4°C. After 3 times wash, plate was then blocked using ELISA/ELISPOT diluent at room temperature for 1 hour. Standard proteins as well as supernatant samples were prepared and 100 μL/well was added to the plate. Each sample was triplicated. Then the plate was sealed and incubated at room temperature for 2 hours. All wells were washed 5 times afterwards. 100 μL/well IL-17A or IFN-γ antibody was then added and incubated at room temperature for 1 hour. After 5 times aspiration, 100 μL/well of diluted Avidin-HRP was added and incubated at room temperature for 30 minutes. Wells were then soaked in wash buffer for 2 minutes prior to aspiration. 100 μL/well of TMB solution was added to each well after aspiration. Plate was incubated for 15 minutes and 50 μL of stop solution was added to each well. Finally plate was read at 450 nm, and concentration of samples was calculated according to the standard curve.Supernatants of lymphocytes pulsed by IRBP antigen complex for 48 hours were harvested and tested for IL-17A and IFN-t-test. A p value of 0.05 or less was considered statistically significant.All experiments were repeated at least three times. Data were expressed as mean ± SEM. The statistical analysis was carried out using Student's p < 0.05), each group containing 5 mice  intraperitoneally from day 0 to day 13 while control group was given with vehicle. On day 14, when inflammation was believed to reach the peak, eyes were harvested for blind histopathological examination. Eyes from control group presented with severe structure destruction, abundant inflammatory cells infiltration both in vitreous body and in retina, vasculitis, photoreceptor layer damage, and moderate to large retinal folds were noticed in almost every case , while 2g 5 mice .p < 0.05).EAU mice immunized using active method undergo priming phase (day 0 to day 6) and effector phase (day 7 to day 13) to develop uveitis. To examine 2ME2 effect on uveitis development in different phases, 2ME2 (15 mg/kg) was intraperitoneally administrated either from day 0 to day 6, from day 7 to day 13, from day 0 to day 13, or vehicle. Compared to control group, HE sections from 2ME2 D0–D13 group remained almost normal retinal structure and exhibited the best curative outcome; those from 2ME2 D0–D6 group had less cell infiltration, milder vasculitis, and less structure disruption, while those from 2ME2 D7–D13 exhibited no much remission in observation . The disp < 0.05). The inhibitory effect of 2ME2 on lymphocyte proliferation attenuated from 2ME2 D0–D13 to 2ME2 D0–D6 and to 2ME2 D7–D13 groups (p < 0.05)  .p < 0.05), each group containing 5 mice (p < 0.05), each group containing 5 mice (γ and IL-17A than those from control after 48 h antigen induction (p < 0.05)  . TogetheThe pathological mechanism during clinical uveitis progression is much more sophisticated than what we have already learnt from EAU models. However, from pathological view, they share lots of similarities including subretinal and choroidal granuloma, retinal vasculitis, retinal detachment, and infiltrating cells both in vitreous cavity and in retina . In thisThe antiproliferative and antimitogenic properties of 2ME2 were widely studied in carcinogenesis and angiogenesis , 21. TheEAU underwent priming phase and effector phase to reach the peak of inflammation. To discover whether the efficacy of 2ME2 was time-dependent, 2ME2 was injected on D0–D6, D7–D13, and D0–D13 separately. Besides the best curative effect in 2ME2 D0–D13 group, priming phase drug employment exerted better outcome than those administrated in effector phase. From these results, we could see that it was more important for 2ME2 to perform its inhibitory effect on priming phase. In priming phase, uveitogenic antigen was processed and presented to T cell, resulting in T cell proliferation. The underlying mechanism of superior efficacy of 2ME2 in priming phase might be related to these processes.In observation of antigen specific lymphocyte proliferation, 2ME2 can significantly inhibit response of IRBP specific lymphocyte harvested from EAU mice. This effect could be maximized in 2ME2 D0–D13 group followed by 2ME2 D0–D6 group, whereas 2ME2 D7–D13 group has the moderate response, only weakened compared to control group. This result is in line with histopathological findings and together proved that a more important effect of 2ME2 is exerted in priming phase. In other researches, 2ME2 can reduce macrophage and NK cells activity, inhibiting T cell activation and proliferation , 25. Havγ and IL-17A, were also downregulated in this process, reducing effector phase T cell mediated target tissue destruction. When activated using costimuli, 2ME2 incubated T cells expressed lower levels of IFN-γ and IL-17A [By isolating lymphocytes from draining lymph nodes and eye-infiltrating cells, T cell differentiation towards Th1 and Th17 lineages was determined to be inhibited by 2ME2 via flow cytometry. Proinflammatory cytokines, IFN-d IL-17A . This isPrevious studies proved that either Th1 or Th17 cells can independently drive specific effector responses during autoimmune diseases development . Targeti2ME2 ameliorated EAU progression and presented a better effect at priming phase. The possible mechanism of this effect could be the inhibitory impact on IRBP specific lymphocyte proliferation and Th1 and Th17 cell differentiation.


GPIHBP1 is an important novel candidate gene for milk production traits. However, the mRNA structure of the bovine GPIHBP1 gene is not fully determined up to now.Identification of functional genes affecting milk production traits is very crucial for improving breeding efficiency in dairy cattle. Many potential candidate genes have been identified through our previous genome wide association study (GWAS). Of these, GPIHBP1 gene. We showed that transcript X5 with a 31 bp insertion and transcript X1 with an 8 bp deletion might have tremendous effect on the protein function and structure of GPIHBP1, respectively. With semi-quantitative PCR and quantitative real-time RT-PCR, we found that the mRNA expression of GPIHBP1, GPIHBP1-X1 and GPIHBP1-X5 in mammary gland of lactating cows were much higher than that in other tissues.In this study, we identified a novel alternatively splice transcript variant (X5) which leads to a 31 bp insertion in exon 3 and also confirmed the other four existed transcripts  of the bovine GPIHBP1 in bovine for the first time and provide useful information for the further functional analyses of GPIHBP1 in dairy cattle.Our study reports a novel alternative splicing of The online version of this article (doi:10.1186/2049-1891-5-50) contains supplementary material, which is available to authorized users. GPIHBP1) is a potential candidate functional gene for milk production traits[GPIHBP1 gene showed strong association with milk yield trait, protein yield and fat percentage with P values 1.02E-10, 1.55E-07 and 6.30E-20, respectively. To confirm the association between the GPIHBP1 gene and milk production traits, we selected a SNP within 5′UTR of GPIHBP1 in another Chinese Holstein population for further association study. This SNP also showed very significant association with milk yield trait, fat percent trait and protein yield trait (unpublished data). Therefore, GPIHBP1 was considered as a novel promising candidate functional gene in dairy cattle.Our previous genome-wide association study (GWAS) in Chinese Holstein population revealed Glycosylphosphatidylinositol-anchored HDL binding protein1 (The GPIHBP1 protein is a glycosylphosphatidylinositol (GPI)-anchored protein of the lymphocyte antigen 6 family. It contains an N-terminal signal peptide, an acidic domain, a lymphocyte antigen 6 (Ly6) domain, and a hydrophobic carboxyl-terminal motif. In the GPIHBP1 is responsible for actively transporting LPL across endothelial cells[GPIHBP1 plays a critical role in the lipolytic processing of triglyceride-rich lipoproteins. Rios et al.[GPIHBP1 gene resulted in extremely high plasma triglyceride and cholesterol level. Beigneux et al.[et al.[GPIHBP1-knockout (GPIHBP1-/-) mice displayed severe hypertriglyceridemia, with a plasma triglyceride level of 1,000-6,000 mg/dL at 7-10 week of age. It was reported that GPIHBP1 was highly expressed in heart and adipose tissue in mice[LPL[GPIHBP1 was the key element for transport and localization of LPL[al cells. Once inal cells, 10. Thuos et al. found thux et al. reportedl.[et al. showed t in mice, 13 and on of LPL, 14, 15 on of LPL.GPIHBP1 remains undetermined yet. The mRNA structure of the bovine GPIHBP1 gene has been keeping on changing in the NCBI database in the most recent years. In the present study, we investigated a new splice variant of bovine GPIHBP1. In order to layout the groundwork for its biological function validation in dairy cattle, we also performed quantitative analysis of the mRNA expression patterns of GPIHBP1 and its novel splice variant in different tissues. We aimed to establish which splice variant is predominantly expressed in bovine tissues.Up to now, the genomic organization of Three Chinese Holstein cows which were in the same period of lactation were selected from Beijing Sanyuan Dairy Farm Center. All of them were fed in a consistent environmental condition. Eight tissues samples  from each cow were collected within 30 min after slaughter and stored at liquid nitrogen. The whole procedure for collection of the tissue samples of all animals was carried out in strict accordance with the protocol approved by the Animal Welfare Committee of China Agricultural University (Permit number: DK996).2O up to 12 μL), 4 μL 5 × First-Strand buffer, 2 μL 0.1 mol/L dTT, 1 μL RNaseout (40U/μL), and 1 μL SuperScrip III RT (200U/μL) . The Mix was heated at 65°C for 5 min and then incubated on ice for at least 1 min. Tubes containing all contents were incubated at 25°C for 5 min, 50°C for 60 min and 70°C for 15 min. To ensure the quality of the first strand cDNA, 1 μL of cDNA was used in a PCR reaction to amplify the glyceraldehyde phosphate dehydrogenase (GAPDH) gene.The total RNA was extracted from the eight tissues of the three cows by using Trizol reagent . The quantity and quality of RNA were measured via an ND-2000 spectrophotometer . Reverse transcription (RT) was carried out in a solution of 20 μL, containing 12 μL Mix (0.5 μL Primer (50 μmol/L) oligio(dt), 0.5 μL Random primer, 1 μL dNTPs (10 mmol/L), 5 μg total RNA and ddHGPIHBP1. The primers  and the Oligo 6.0 software. For each amplicon, 1 μL of cDNA , 2.0 μL of 10× PCR buffer, 250 μmol/L of each dNTP, 0.5 units of HotstarTaq polymerase , and 0.5 μmol/L of primer (Life Technologies) were used in a total 20 μL reaction. The reaction was denatured for 10 min at 95°C, then 35 cycles of 94°C for 30 s, special annealing temperature for 30 s and 72°C for 30 s, and a final extension of 72°C for ten min. The products were electrophoresed on 2% agarose gels and stained with ethidium bromide.PCR reactions were performed to amplify the coding regions of The purified double-stranded DNA  was cloned in pMD18-T . The products of the ligation reactions were transformed into competent cells. Twenty colonies per sample were selected randomly for sequencing. With the DNAMAN 7.0 software, we performed multiple sequences alignment analysis.http://tcoffee.vital-it.ch/apps/tcoffee/do:regular) was used to align amino acid sequences of the bovine and human GPIHBP1 proteins. We predicted the open reading frame of the bovine GPIHBP1 transcript X5 using ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/). Secondary structures of the GPIHBP1 proteins were predicted using the PSIPRED v3.3 website tool (http://bioinf.cs.ucl.ac.uk/psipred/). SignalP 4.1 [http://mendel.imp.ac.at/gpi/gpi_server.html)[http://swissmodel.expasy.org/)[http://swissmodel.expasy.org/)[The T coffee website tool  was useder.html) was utiler.html), was usesy.org/). The repsy.org/).2+ (50 mmol/L), 0.5 μL dNTPs (10 mmol/L), 0.5 μL of each primer  was performed on the eight tissues of three cows. TaqMan Real-time PCR assays were performed using 7500Fast . The PCR amplification mix consisted of 2 μL 10× PCR Buffer, 1.2 μL MgT method.GPIHBP1 in samples of mammary gland. After PCR amplification with primer 1 and primer 3, we observed three  have the same translation initiation site.Currently, four transcript variants  of http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to predict all possible ORF of transcript variants X5. As a result, five potential ORF were predicted which had initiation codon and termination codon. The amino acid sequences corresponding to the five ORF were obtained using the DNAMAN 7.0 software and named bovine GPIHBP1 P5.1, P5.2, P5.3, P5.4, and P5.5, respectively  and encode a 171-amino acid protein that was named bovine GPIHBP1 P2. In contrast, the transcript variant X1 contains a different ORF and encodes a 142-amino acid protein, which named bovine GPIHBP1 P1. However, the ORF of transcript X5 was still not known clearly up to now. The ORF Finder software  using Big-PI Predictor[β-sheet structures. However, due to the low alignment quality between target and specified template, the tertiary structures of the predicted bovine GPIHBP1 P5.1, P5.2, P5.3, P5.4, and P5.5 could not be constructed.The predicted secondary structures of the bovine GPIHBP1 amino acid sequences were compared with that of the human GPIHBP1 protein. The  Figure A. By usi Figure A, B. Theredictor, 22  and multiple comparisons were conducted with R software. We found that mRNA expression level of GPIHBP1, GPIHBP1-X1 and GPIHBP1-X5 all were significantly different among eight tissues (P < 0.05), in which the mRNA expression levels were significantly higher in mammary gland than other tissues (P < 0.05). And all of GPIHBP1-X1, GPIHBP1-X5 and overall GPIHBP1 had much lower expression level in liver, kidney and muscle  expression pattern in bovine mammary gland at different stages of lactation was quite similar to the lactation curve, which suggest that LPL is important for maintenance of milk synthesis[GPIHBP1 served as the transporter and the platform for the lipoprotein lipase-mediated lipolysis processing[GPIHBP1 is essential for LPL to realize its biological function and play an important role in the process of producing milk fat and maintenance of milk synthesis.Tissue-specific mRNA expression patterns are important for revealing functional candidate genes associated with milk production traits. The spe in mice, 13. Pre in mice–26 showeynthesis. Meanwhiocessing. TherefoCommonly, alternative splicing may change the structure of transcripts of a gene and the protein encoded by the gene, leading to profound functional alternation. It has been demonstrated that alternative splicing could affect the binding properties, intracellular localization, enzymatic activity, protein stability and post-translational modifications of a large number of proteins. The effGPIHBP1 gene. The proteins of transcripts X2, X3 and X4 have the classical structure of the GPIHBP1 protein consisting of the N-terminal signal peptide, UPAR-Ly6 domain and C-terminal GPI-Modification Site. The protein P1 encoded by transcript X1 has the UPAR-Ly6 domain and the C-terminal GPI-Modification Site, but it lacks the signal peptide and acidic domain. It is not clear if this protein would be ever produced and secreted because of lacking the signal peptide. However, even if it is at all secreted as normal, it is also a non-functional GPIHBP1 because it lacks acidic domain, which makes it unable to bind to LPL[GPIHBP1 in mammary gland of dairy cattle by nonsense-mediated decay and thus affect milk production traits indirectly.In this study, we identified that there were five transcripts  of the bovine d to LPL. The spld to LPL. TherefoGPIHBP1 gene. We identified a novel alternatively spliced transcript variant of GPIHBP1 gene (GPIHBP1-X5) with 31 bp insertion in the exon and also confirmed other four existed transcripts  of the GPIHBP1 in Chinese Holstein cow. And we found that the 8 bp deletion of transcript X1 and 31 bp insertion of transcript X5 have tremendous effect on protein function and structure, respectively. Based on the results of Taq-Man RT-PCR, we found that GPIHBP1-X1, GPIHBP1-X5 and GPIHBP1 expressed in higher level in mammary gland than in other tissues of lactating dairy cow. In conclusions, our findings provided more information for the further functional analyses of GPIHBP1 in dairy cattle.This study is the first report on alternative splicing of bovine Table S2. Sequences of forward (F) and reverse (R) primers and probes used for TaqMan real time PCR. Table S3. Specific primers for confirming the existence of 8 bp deletion and 31 bp insertion. Table S4. The amino acid sequences of five potential open reading frames of GPIHBP1 X5. Table S5. Predicted GPI-modification sites for amino acid sequence of GPIHBP1 splice variants. (XLSX 15 KB)Additional file 1: Table S1: Sequences of forward (F) and reverse (R) primers for PCR amplification of the coding region of GPIHBP1.


Azide-alkyne Huisgen “click” chemistry provides new synthetic routes for making thermoplastic polytriazole polymers—without solvent or catalyst. This method was used to polymerize three diester dialkyne monomers with a lipid derived 18 carbon diazide to produce a series of polymers  free of residual solvent and catalyst. Three diester dialkyne monomers were synthesized with ester chain lengths of 4, 9, and 18 carbons from renewable sources. Significant differences in thermal and mechanical properties were observed between C18C9 and the two other polymers. C18C9 presented a lower melting temperature, higher elongation at break, and reduced Young's modulus compared to C18C4 and C18C18. This was due to the “odd-even” effect induced by the number of carbon atoms in the monomers which resulted in orientation of the ester linkages of C18C9 in the same direction, thereby reducing hydrogen bonding. The thermoplastic polytriazoles presented are novel polymers derived from vegetable oil with favourable mechanical and thermal properties suitable for a large range of applications where no residual solvent or catalyst can be tolerated. Their added potential biocompatibility and biodegradability make them ideal for applications in the medical and pharmaceutical industries. Dwindling reserves and price increases are driving research for alternatives to petrochemical feedstocks used in materials and fuels. Polymers derived from nonpetrochemical sources are becoming more viable and economical as improvements in feedstock, processing, and technology are developed. One area receiving significant attention is that of polymers derived from vegetable oils. Vegetable oils are abundant, renewable, and possess favourable properties including biofragmentability and biocompatibility compared to petrochemical analogs . They caEnvironmentally favourable chemistries using benign conditions and producing little or no byproducts are the subject of extensive research. Polymers are generally synthesized using solvents and metal catalysts. Although tolerated at small levels for commodity polymers, some solvents, such as DMF and 1,2-dichlorobenzene, and metal catalysts, are toxic and potentially carcinogenic and are very hard to remove. This is particularly critical in biomedical and pharmaceutical applications where solvent and catalyst free synthesis routes are highly preferred . More geRecently, azide-alkyne Huisgen 1,3-dipolar cycloaddition has begun to find its way into the repertoire of tools available to green chemists . This re2. It is a common commercial feedstock and can be used to produce industrially important products, such as 1,4-butanediol and polyesters [The goal of this work was to create lipid derived thermoplastic polymers using the commonly available and economical feedstocks: succinic acid, azelaic acid, and octadec-9-enedioic acid—all of which are produced from renewable resources . Succinilyesters . Worldwilyesters , 19. Botlyesters . Octadeclyesters , 22. TheThe intent of varying the chain length of the monomer was to establish the degree of dependence on monomer chain length and degree of asymmetry. Furthermore, the relationship between even and odd chain length monomers, known as the odd-even effect, can be probed by comparing the properties of the azelaic acid based monomer (9 carbons) with the two other monomers, each having an even number of carbon atoms. Polyesters with even carbon numbered monomers demonstrate higher melting temperatures and enthalpies of fusion than their odd carbon number analogues , 24. in situ and further modify the unsaturation to afford a potentially biocompatible polymer with easily tunable functionalities.In the present work, three thermoplastic polytriazoles from diester dialkynes and a diazide monomer derived from oleic acid  were synthesized and characterized. The numbers of carbon atoms in the diester dialkyne monomers are 4, 9, and 18, with 18 for the diazide monomer. This work presents a new route to the creation of melt-processable polymers from renewable vegetable oils without residual solvent or catalyst. Furthermore, because of the presence of unsaturated sites in the monomers, it may be possible to perform polymerization on a suitable substrate4 (95%), N,N′-dicyclohexylcarbodiimide (DCC) (99%), and 4-dimethylaminopyridine (DMAP) (99%) were purchased from Sigma-Aldrich and used as received. (E)-octadec-9-ene-1,18-diol and (E)-1,18-octadec-9-endiacid were synthesized using a previously reported procedure [Azelaic acid (98%), oleic acid (85%), sodium azide (99%), propargyl alcohol (99%), 4-toluenesulfonyl chloride (98%), triethyl amine (99%), succinic acid (99%), Grubbs catalyst 2nd generation, LiAlHrocedure , that isE)-1,18-octadec-9-endiacid (C18), azelaic acid (C9), and succinic acid (C4) through either Steglich or Fischer esterification -Octadec-9-ene-1,18-diyl bis(4-methylbenzenesulfonate)   (1). (E)-Octadec-9-ene-1,18-diol  was added to a round bottom flask kept in an ice bath (0°C). 4-Toluenesulfonyl chloride  dissolved in 150 mL of CH2Cl2 and was added dropwise, then 0.30 g (2.5 mmol) of DMAP and 12.0 g (119 mmol) of triethylamine dissolved in 20 mL CH2Cl2 was added dropwise. The reaction was allowed to warm to room temperature and proceeded until complete consumption of the starting material, as determined by TLC . The contents of the flask were diluted with 150 mL CH2Cl2 and washed with water (3 × 150 mL), 2.5 N HCl solution (3 × 150 mL), 4% aqueous sodium bicarbonate (3 × 150 mL), and finally saturated NaCl brine (3 × 150 mL). The organic layer was dried with anhydrous Na2SO4 and concentrated under reduced pressure yielding 22.3 g of slightly yellow crystal. The solid product was purified by recrystallization from hexanes twice and dried in a vacuum oven. 18.0 g of a white solid was recovered (72% yield). 1H-NMR (500 MHz in CDCl3): δ 1.21 , 1.62 , 1.94 , 2.45 , 4.01 , 5.36 , 7.34 , and 7.79 .(E)-1,18-Diazidooctadec-9-ene (2). 8.2 g of (1) was added to a round bottom flash equipped with a reflux condenser. 90 mL of DMSO was added and 12.0 g of NaN3 dissolved in 10 mL deionized water was introduced with stirring. Immediately following addition, the reaction turned slightly yellow and was warm to the touch. It was heated to 100°C and stirred for 12 hours at this temperature. Upon completion, the mixture was cooled to room temperature and poured into 250 mL of ice water to precipitate the product and separate the DMSO into the aqueous phase. The organic layer was extracted with ethyl acetate (3 × 150 mL) and washed with water (2 × 150 mL) and NaCl brine (2 × 150 mL). The organic phase was dried over Na2SO4 and concentrated under reduced pressure. It was then purified by column chromatography on silica gel using a 50 : 1 ratio of hexanes to ethyl acetate to afford a colourless liquid . 1H-NMR (500 MHz in CDCl3): δ 1.29 , 1.59 , 1.96 , 3.25 , and 5.38 .(E)-Di(prop-2-yn-1-yl) octadec-9-enedioate (3). (E)-1,18-octadec-9-ene diacid  was added to a round bottom flask with 200 mL of chloroform and heated to 50°C. The mixture was stirred until it became clear and then 7.89 g propargyl alcohol (140 mmol) was added to the flask. N,N′-dicyclohexylcarbodiimide  in 50 mL CHCl3 was added dropwise with 0.5 g 4-dimethylaminopyridine (DMAP) over 30 min. A reflux condenser was then equipped and the solution was stirred for 12 hours at 50°C then cooled to room temperature and filtered over celite to remove precipitated dicyclohexylurea. The chloroform was removed under reduced pressure and the compound was dissolved in hot hexanes and filtered while hot over celite. The product was recrystallized from hexanes twice, with only the trans product recovered and dried in a vacuum oven overnight to yield 20.4 g of a white powder (yield: 82%). 1H-NMR (500 MHz in CDCl3): δ 1.31 , 1.65 , 1.97 , 2.36 , 2.47 , 4.68 , and 5.38 .Di(prop-2-yn-1-yl) nonanedioate  (4). Azelaic acid  was added to a round bottom flask with 75 mL toluene and heated to 100°C. The mixture was stirred until the solution became clear then 20 mL (347 mmol) of propargyl alcohol was added. Five drops of concentrated sulfuric acid in 2 mL toluene was slowly added to the mixture and the solution was stirred for 12 hours. The reaction was cooled to room temperature and then it was diluted with an additional 75 mL toluene and washed with water (3 × 100 mL), then a sodium bicarbonate solution, (3 × 100 mL), and finally saturated NaCl brine (2 × 100 mL). The organic layer was dried with anhydrous sodium sulfate and concentrated under reduced pressure. The product was purified by vacuum distillation at 150 mtorr and the fraction boiling between 133°C and 136°C collected as a colourless liquid . 1H-NMR (500 MHz in CDCl3):δ 1.32 , 1.63 , 2.35 , 2.47 , and 4.67 .Di(prop-2-yn-1-yl) succinate  (5). Succinic acid  was added to a round bottom flask with 75 mL toluene and heated to 100°C. The mixture was stirred until the solution became clear and 20 mL propargyl alcohol (357 mmol) was added. 5 drops of concentrated sulfuric acid in 2 mL toluene was then slowly added to the mixture, and the solution was stirred for 12 hours. The reaction was cooled to room temperature, then the mixture was diluted with an additional 75 mL toluene and washed with water (3 × 100 mL), then a sodium bicarbonate solution (3 × 100 mL) and finally with saturated NaCl brine (2 × 100 mL). The organic layer was dried with anhydrous sodium sulfate and concentrated under reduced pressure. The product was purified by vacuum distillation at 160 mtorr and the fraction boiling between 89°C and 91°C was collected as a colourless liquid . 1H-NMR (500 MHz in CDCl3): δ 2.48 , 2.69 , 4.69 .Equal molar ratios of a dialkyne monomer and a diazide monomer were added to a polytetrafluoroethylene (PTFE) round bottom flask. The flask was purged with nitrogen for 15 minutes then placed in an oil bath under nitrogen protection and heated from room temperature to 110°C gradually over a 2-hour period to prevent thermal decomposition of the reactants in the exothermic polymerization. It was then stirred for an additional 20 hours at this temperature. The polymer product was cooled to room temperature and cast into films by melting at 170°C inside a stainless steel mold (70 × 30 × 0.6 mm) covered with a sheet of PTFE. The molten sample was pressed on a Carver 12-ton hydraulic heated bench press . Three metric tons of pressure were applied isothermally for 15 min to the mold. The sample was then cooled to 20°C over 30 min by pumping coolant through the plates on the hydraulic press period using a Julabo temperature controlled circulating bath . The polymer film was stored in a sealed container until analysis.1H spectra were recorded on a Varian Unity-INOVA at 499.695 MHz. 1H chemical shifts were internally referenced to CDCl3 (7.26 ppm) for spectra recorded in CDCl3 and 7.19 ppm for spectra recorded in 1,2-dichlorobenzene-d4. All spectra were obtained using an 8.6 μs pulse with 4 transients collected in 16,202 points. Datasets were zero-filled to 64,000 points and a line broadening of 0.4 Hz was applied prior to Fourier transforming the sets. The spectra were processed in ACD Labs NMR Processor, version 12.01.The 1H NMR of the monomers, vinylic protons −CH=HC= present around δ 5.38 ppm and allylic −C=CH−CH2− at δ 1.97. In the alkyne monomers, the propargylic proton −C(=O)OCH2C≡CH appears at δ 2.47-2.48 ppm, and the −C(=O)OCH2C≡CH appears at δ 4.67–4.69 ppm. The ester −CH2C(=O)O− occurs at δ 2.35-2.36 for the C9 and C18 alkynes but is shifted to δ 2.69 ppm in the C4 monomer due to the close proximity of the ester groups to each other. In the tosylate intermediate, shifts at δ 7.34 ppm and δ 7.79 ppm both correspond to different sets of protons on the aromatic ring and the benzylic −Bz−CH3 presents at δ 2.45. The tosylate ester influences both the −CH2OS− and −CH2CH2OS−, with shifts appearing at δ 4.01 and 1.62 ppm, respectively. Furthermore, in the azide monomer, we see a disappearance of the indicative shifts of the tosyl group. The −CH2N3 presents at δ 3.25 ppm and –CH2CH2N3 at 1.59 ppm. The remaining alkane −CH2CH2CH2- protons fall between δ 1.21 and 1.45 ppm.In a typical Thermogravimetric Analysis was conducted on a TGA Q500 . The sample (10–20 mg) was heated from room temperature to 600°C at a rate of 10.0°C/min under a nitrogen flow of 60.0 mL/min. Calorimetry studies were performed following ASTM E1356 standard on a DSC Q200  equipped with a refrigerated cooling system. The sample (5-6 mg) was initially heated to 180°C for 10 min to remove thermal history and then cooled at a rate of 5.0°C/min down to −80°C, where it was held isothermally for 5 min. Finally the sample was heated from −80°C to 180°C at a rate of 5.0°C/min.μm.Dynamic mechanical analysis (DMA) measurements were carried out following ASTM D7028 standard on a Q800  analyzer equipped with a liquid nitrogen cooling system. The sample was cut into a rectangular shape (17.5 mm × 12.0 mm × 0.60 mm) and was analyzed in the single cantilever bending mode. It was heated at a constant rate of 1.0°C/min from −100°C to 40°C. The measurements were performed at a frequency of 1 Hz and fixed oscillation displacement of 15 Tensile tests were performed according to ASTM D882 standard using a mechanical analyser  equipped with a 25-kg load cell. The film was die-cut by an ASTM D638 type V cutter. The sample was stretched at a rate of 60 mm/min from a gauge of 35 mm at room temperature.−1 for 128 repeated scans at a spectral resolution of 0.4821 cm−1. 16 repeated scans were used for monitoring the polymerization. The data was background corrected and baselined by the machine software  and exported in spreadsheet format for graphing.ATR-FTIR spectra were collected using a Thermo Scientific Nicolet 380 FTIR spectrometer  fitted with a PIKE MIRacle attenuated total reflectance (ATR) system . 0.60 mm thick polymer samples were placed onto the ATR crystal and pressed by a mechanical arm. Spectra of the polymer films were acquired over a scanning range of 500–4000 cmα radiation source (λ = 1.540598 Å) and a PIXcel3D detector. The scanning range was 2.0062°–30.000° (2θ) with a step size of 0.0263°. Data analysis was performed using PANalytical's X'Pert HighScore 3.0.4 software. The relative degree of crystallinity was estimated according to a well-established procedure [XC) is calculated byAC is the area under the resolved crystal diffraction peak contribution and AA is the area of the amorphous contribution.The crystalline structure and relative crystallinity of the polymers were examined by wide-angle X-ray diffraction (WAXD) using an EMPYREAN diffractometer system  equipped with a filtered Cu–K rocedure . The pern = 4, 9, and 18 carbon atoms were transformed into a dialkyne terminated diester monomers. The length of the dialkyne was varied by choosing three economically priced and plant derived dicarboxylic acids: succinic acid (C4), azelaic acid (C9), and octadec-9-enedioic acid (C18). The choice of ester linkages was made because they are simpler to synthesize from carboxylic acids, which are readily manufactured from vegetable triglycerides on a commercial scale. Ester linkages have also been shown to add biodegradability to polymers [E)-1,18-diazidooctadec-9-ene, which was prepared from the same C18 diacid, has 18 carbon atoms separating two high energy (azide) nitrogen groups. The significance of this is that the ratio of 3 carbon to 1 nitrogen atoms fulfills an important safety consideration for azides. This ratio is generally accepted as sufficient to prevent the rapid decomposition or explosion of azide groups and to allow safe storage and processing [Vegetable derived dicarboxylic acids with polymers . The diaocessing .3, CH2Cl2, THF, 1,2-dichlorobenzene, N-methyl-2-pyrrolidone, methanol, 2-propanol, diethyl ether, ethyl acetate, acetone, hexanes, and acetonitrile.In solvent polymerization, the solvent can aid in dissipating heat of exothermic events, which was not possible in this system. To circumvent this issue, the monomers were gradually oligomerized by heating to the polymerization temperature (110°C) over 2 hours with stirring. The viscosity increased gradually as oligomerization proceeded allowing prolonged mixing (which did not occur with rapid heating). Characterization of the polymer molecular weights was not possible due to poor solubility in common solvents, including DMF, DMSO, CHCl1H NMR of the polymers in 1,2-dichlorobenzene-d4 was run at 45°C for improved polymer solubility. This temperature was a good compromise between increasing solubility and thermal noise. 1H NMR spectrum of C18C9, representative of the C18Cn polymers, is shown in http://dx.doi.org/10.1155/2014/792901). The limited solubility of the polymer caused significant line broadening of the NMR peaks, but the characteristic peaks for both the 1,4-(at δ 7.47) and the 1,5-substituted 1,2,3-triazoles (at δ 7.62) were observed in all the polymer samples. As can be seen in H2N3, δ 3.25) or alkyne  groups on the monomers, indicating complete conversion. The ratio of the 1,4- to the 1,5- substitution estimated by integrating the area of each triazole C–H peak was 2 : 1.N=N=N)   [Selected ATR-FTIR spectra obtained during the monitoring of the polymerization of C18C18 are shown in 1 in −1)  appeared−1 in all the polymers represents the free ester carbonyl C=O stretch. Only one peak representing the ester carbonyl C=O stretch is visible in the FTIR of C18C18 and a second peak was observed at 1730 cm−1 and 1720 cm−1 for C18C9 and C18C4 , recrystallization temperatures , and peak temperatures of melting . However, peak temperature of the second melt was relatively higher in C18C9 (TM2 = 86.5 ± 0.1°C), compared to C18C18 (TM2 = 73.5 ± 1.2°C). The enthalpy of the first and second endotherms in C18C9 was 6.0 ± 0.8 and 14.1 ± 0.2 J/g, respectively, and in C18C18 was 3.0 ± 0.3 J/g and 30.5 ± 0.9 J/g, respectively. The peak temperature of the two endotherms in C18C4 were at 97.5 ± 1.4 and 121.3 ± 1.5°C; significantly higher than in C18C9 and C18C18.The DSC heating profiles (5°C/min) of the polymers, followed by the subsequent cooling (5°C/min), are shown in TM1) corresponds to the portion which has recrystallized. The enthalpy of the second endotherm was roughly equal to the enthalpy measured for the exotherm obtained during the crystallization from the melt (cooling rate of 5°C/min) in each polymer  and 3.91 Å (010), characteristic of the monoclinic subcell, similarly to what was reported for vegetable oil derived poly (ester-urethanes) . C18C9 pTGA and DTG curves of the polymers are shown in Figures α-carbon atoms between the esters [The first decomposition peak in the DTG  for C18Ce esters . This maδ of the polymers are shown in Figures δ versus temperature curves indicated homogeneous systems with single glass transitions. The prolonged glass transition observed by DSC is probably due to enthalpy of relaxation effects. The first peak observed at ~−85°C is attributable to the so-called β transition. As clearly seen in the loss modulus graph in β-transitions in C18C4 were much more intense than C18C9 and C18C18 where it was very weak. This transition may be attributed to the limited mobility of the ester carbonyl group, or the shorter methylene chain segments, both of which increase through the series from n = 4 to 18 [n; as the chain length increased, its mobility would decrease.The viscoelastic properties of the polytriazoles were studied using DMA. The loss modulus, storage modulus, and tan  4 to 18 , 43. ThiTg of C18C18 and C18C9, as determined from the peak tan δ curve, Tg as measured by DMA can be explained in terms of the structural differences between the polymers and related to the competing effects of the hydrogen bonding introduced by the triazole groups and the esters contributions which in fact determine a “hydrogen bond density and distribution”. Note that the trends in the Tg obtained by DMA in Tg difference between C9 and C18.In E and XC, the two parameters are strongly correlated. This is not surprising as the effect of crystallinity on polymers Young's moduli is well known [Stress-strain curves of the polymers are shown in ll known . All thrThe elongation at break of the less crystalline C18C9 was 196.6 ± 16.5%: much higher than C18C4 at 39.6 ± 3.8% and C18C18 at 10.7 ± 3.8% . This reAs the predominating hydrogen bonding mechanism shifts from triazole self-association to triazole—carbonyl on shortening of the diester chain segment, a transition in properties is observed, with C18C9 presenting mid-way characteristics. FTIR data suggests that the hydrogen bonding from triazole-triazole and triazole-ester hydrogen bonding is reduced in C18C9 compared to C18C18 and C18C4. The crystallinity of the C18C9 polymer was also significantly reduced. Previous studies have shown that odd-carbon length monomers can prevent up to 50% of the hydrogen bonding interactions due to altered orientations . An odd Three novel thermoplastic polytriazoles were produced by uncatalyzed and solvent free polymerization of vegetable oil derived diester alkyne and diazide monomers. The three dialkynes were synthesized from economically priced succinic acid (C4), azelaic acid (C9), and oleic acid (C18). The diazide monomer, prepared from C18 diacid, had a ratio of 3 carbon to 1 nitrogen atoms; generally accepted as sufficient to prevent the rapid decomposition or explosion of azide groups and to allow safe storage and processing.The monomers and polymers were fully characterized by NMR and ATR-FTIR. Crystal structure, thermal behavior, and mechanical properties of the polymers, measured using XRD, TGA, DSC, DMA, and tensile test, were related to the structural details of the repeating unit, compared and contrasted to the paper. The trends observed in the thermal behaviour and mechanical properties of the polymers were explained based on differences in their crystallinity and ability to hydrogen bond. The polymers synthesized from even chain length monomers had significantly higher crystallinity, higher melting points and melt enthalpy, higher Young's modulus values, and lower elongation-to-break percentages when compared with the odd chain length monomer. The crystallinity and melt and crystallization enthalpies of C18C9 were approximately half of those of the other polymers. The odd-even effect, which dictates ordering of groups in symmetrical or asymmetrical configurations based on the monomer chain length, was used to explain this behaviour. The differences observed in physical property between the polymers are consistent with the presence of two different hydrogen bonding systems: hydrogen bonding between ester—triazole groups, and triazole—triazole groups. C18C18 was suggested to have predominantly ester—triazole hydrogen bonding, C18C4 predominantly triazole—triazole hydrogen bonding and the third polymer, C18C9, which has an odd carbon length monomer, a mixture of these two hydrogen bonding systems. This work advances effective routes for “green” polymerization and provides a catalyst and solvent free method to make functional thermoplastic polymers from vegetable oils. This opens the possibility of future use in applications sensitive to catalyst or solvent contamination, including medical and pharmaceutical applications.A magnified proton NMR spectra of the polymer C18C9 from Figure 1, showing enhanced resolution of the triazole region shifts is provided in Figure S1 in the Supplementary Material.


Accurate and reliable assessment of human epidermal growth factor receptor type 2 (HER2) status is important for selecting patients with gastric cancer who may benefit from trastuzumab treatment. Here we examined the impact of formalin fixing conditions on HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in xenografted tumor tissues.Xenografted tumor tissues of the human gastric cancer cell lines NCI-N87, SCH, and SNU-16 were collected and kept at room temperature for 0, 6, or 24 h before being fixed with 10 % neutral buffered formalin (NBF) for 24 h or 5, 7, or 10 days and embedded in paraffin. Use of 10 % NBF, 20 % NBF, or nonbuffered formalin as fixative was investigated.The HER2 IHC scores for NCI-N87, SCH, and SNU-16 tumors were 3+, 2+, and 1+, respectively, when specimens were fixed with 10 % NBF for 24 h immediately after resection of the tumors. Specimens left for longer than 6 h before fixation had shrinkage of the tumor periphery and decreased immunostaining intensity in this region in all specimens. In SCH and SNU-16 specimens, starting fixation 24 h after tumor tissue collection induced autolysis and reduction of the number of stained cells, and 10-day-fixation lowered the HER2 score. Prolongation of fixation time did not affect FISH results, but if samples were left for more than 6 h before fixation, the FISH score was strongly reduced in SCH specimens (2.3 to 1.3). Reduced IHC staining intensity was observed with 20 % NBF and nonbuffered formalin compared to 10 % NBF.The time to and length of fixation of tumor specimens can affect HER2 IHC and FISH scores. The fixative used can affect IHC results. Gastric cancer is the forth frequently diagnosed tumors in the world with 989,000 cases estimated to have occurred  and is tThe ToGA trial, an open-label, international, phase III, randomized controlled trial, demonstrated that patients with HER2-positive gastric cancer assigned to receive trastuzumab plus standard chemotherapy had significantly longer overall survival (OS)  compared with patients with HER2-positive gastric cancer assigned to receive chemotherapy alone [Immunohistochemistry analysis, which evaluates protein expression, and FISH analysis, which evaluates gene amplification, are major methods for HER2 testing. Although assessing HER2 status accurately and reliably is of great importance to determine whether patients have HER2-positive breast or gastric cancer, there is variability of sample preparation for HER2 testing . A numbeBy virtue of the ASCO/CAP guidelines, the method of preparing specimens has been standardized in breast cancer. In Japan, HER2 testing guidelines for gastric cancer were defined by the trastuzumab pathological advisory board for gastric cancer in line with ASCO/CAP guidelines. Paraffin sections are prepared, and the IHC scoring system is used according to these criteria that aimed to develop a validated HER2 scoring system for gastric cancer . HoweverFive-week-old male BALB-nu/nu mice (CAnN.Cg-Foxn1<nu>/CrlCrlj nu/nu) were obtained from Charles River Laboratories Japan . All animals were housed in a pathogen-free environment under controlled conditions . Chlorinated water and irradiated food were provided ad libitum. All animals were allowed to acclimatize and recover from shipping-related stress for 1 week before the study. The health of the mice was monitored by daily observation. All animal experiments were conducted in accordance with the Guidelines for the Accommodation and Care of Laboratory Animals promulgated in Chugai Pharmaceutical Co., Ltd.2. The histological type of each cell line is shown in Table Three human gastric cancer cell lines were used in the present study. NCI-N87 and SNU-16 cells were purchased from American Type Culture Collection (ATCC) . SCH cells were purchased from Japan Health Sciences Foundation . NCI-N87, SNU-16, and SCH were maintained in RPMI-1640  supplemented with 10 % (v/v) fetal bovine serum at 37 °C under 5 % CO6 cells/mouse of a human gastric cancer cell line, either NCI-N87, SCH, or SNU-16. Tumor xenograft tissues were collected and allowed to stand at room temperature for 0, 6, or 24 h before being fixed with 10 % neutral buffered formalin (NBF) for 24 h, or 5, 7, or 10 days, and then embedded in paraffin. To compare the effect of fixing solution on HER2 IHC, 10 % NBF, 20 % NBF, 10 % nonbuffered formalin, or 20 % nonbuffered formalin was used. Specimens were prepared from two separate tumors from mice inoculated with NCI-N87 and SNU-16 and three tumors from SCH-inoculated mice.Each mouse was inoculated subcutaneously into the right flank with 5 × 10HER2 protein expression and HER2 gene amplification in tumors were examined by IHC using HercepTest  and by FISH using PathVysion HER2 DNA Probe , respectively, at SRL  as described previously . HistoloThree tumor tissues with different HER2 status were used in this study. The HER2 status of these tumor tissues were assessed by IHC and FISH, using specimens fixed with 10 % NBF for 24 h immediately after resection of the tumors. The IHC scores for NCI-N87, SCH, and SNU-16 were 3+, 2+, and 1+, respectively, and HER2/CEP17 ratios by FISH were 8.4, 2.3, and 1.4, respectively  0 and 24 h, (2) 0 h and 10 days, (3) 6 and 24 h, (4) 24 and 24 h, or (5) 24 h and 10 days were assessed by FISH (HER2/CEP17 ratio). The mean of the HER2/CEP17 ratios of specimens of times (1), (2), (3), (4), and (5) were 2.3, 2.6, 1.3, 1.2, and 1.1, respectively, suggesting that prolonged fixation time did not affect the FISH results but that delay to fixation strongly reduced the FISH score  because, in clinical specimens, those judged as HER2 IHC score 2+ are considered to be equivocal and the FISH test is recommended to make a final diagnosis for trastuzumab application. Therefore, it makes sense to examine whether fixative type affects the results of the HER2 IHC test in tumor tissue specimens of HER2 IHC score 2+. For this purpose, we used 10 % NBF, 20 % NBF, 10 % nonbuffered formalin, or 20 % nonbuffered formalin. The tumor tissues were fixed with each fixative immediately after collecting. Fixing with 20 % NBF, or 10 % or 20 % nonbuffered formalin for 24 h reduced the staining intensity of HER2 and reduced the HER2 IHC score from 2+ to 1+ in one of three specimens compared to fixing with 10 % NBF Fig . When tuIn breast cancer, ASCO/CAP issued guidelines to standardize fixation for increased HER2 testing accuracy. In addition, many investigations into the optimal conditions for accurate HER2 testing have been conducted –18. DelaIn the present study, leaving specimens at room temperature for more than 6 h before fixation led to shrinkage at the tumor periphery and decreased the staining intensity at the shrinkage area in all the tumors assessed, irrespective of HER2 status. However, a decrease in HER2 IHC score was observed only in SCH specimens with a 24-h delay to fixation, whereas the HER2/CEP17 ratio of FISH in SCH was decreased from 2.3 to 1.3 if the tumor sample was left at room temperature for only 6 h before fixation. In breast cancer, it has been reported that a delay to formalin fixation affects FISH results but not IHC scores . TherefoWith regard to fixation time, prolonged fixation did not affect the HER2 IHC scores of NCI-N87 tumors (HER2 3+ score)  was reduced. We also examined the effect of fixatives in NCI-N87 (IHC score 3+); however, there were no differences in staining between fixatives. Staining of specimens that moderately express HER2 protein may therefore be affected more easily by the type of fixative used compared to staining of specimens with high expression of HER2. In the present study, we did not examine the effect of fixative type on FISH results, because, for gastric cancer, IHC testing is recommended as a first examination for HER2, followed by confirmative testing by FISH for IHC score 2+ specimens.In the clinical setting, it is important to take unique HER2 staining characteristics into consideration when standardizing HER2 testing in gastric cancer. One such characteristic is the heterogeneity defined as less than 30 % of tumor cells staining positive or only focal staining of tumor cells , 19. GasIn conclusion, our results indicate that delay to fixation and length of fixation may affect the HER2 IHC score in a gastric cancer model. Nonbuffered formalin or high concentrations of neutral buffered formalin (NBF) may also affect IHC results. These results highlight that it is of critical importance to optimize sample preparation conditions for HER2 testing in gastric cancer.


Recent investigations have highlighted that therapeutic artificial microRNAs could be promising candidates for cancer therapy through the modulation of tumor promoter or suppressor. MEK kinase 1 (MEKK1) is expressed by mitogen-activated kinase kinase kinase 1 (MAP3K1), an important kinase that links Ras activation to MAPK signaling. In the present study, we showed that synthetic MAP3K1-targeting artificial miRNA may provide considerable beneficial effects in the prevention of breast cancer growth and metastasis. We showed that MEKK1 was highly expressed in human breast cancer specimens, compared with adjacent normal tissues. Using a miRNA-expressing lentivirus system, we delivered a artificial miRNA (Map3k1 amiRNA) that targets MAP3K1 into 4T1 breast cancer cells and investigated the impact of MAP3K1-targeting miRNA on the growth and invasive behavior of breast cancer in vitro and in vivo. We found that overexpression of Map3k1 amiRNA led to impaired activities of p-ERK and p-p38. In addition, Map3k1 amiRNA induced marked proliferative impairment and invasive attenuation in breast cancer cells. However, Map3k1 amiRNA did not have evident influence on the apoptotic response of 4T1 cells. Moreover, using in vivo nude mice model, we identified that Map3k1 amiRNA attenuated tumor growth and lung metastasis of breast cancer cells. Taken together, our findings explicitly indicated that MEKK1 exerted important oncogenic property in breast cancer development, and MAP3K1-targeting artificial miRNA may provide promising therapeutic effects in the treatment of breast cancer. Breast cancer represents the most frequent cancer among women and a leading cause of cancer-associated mortality around the world. It is estimated that there are annually 1.7 million breast cancer cases worldwide, with 522,000 deaths  in the regulation of cellular signaling and cancer biology  according to the manufacturer’s instruments.Murine breast carcinoma 4T1 cells and Human breast cancer cell lines, MCF-7 and MDA-MB-231 were purchased from ATCC. The cells were maintained in RPMI-1640 medium (Gibco) supplemented 10 % fetal bovine serum (Hyclone). Cells were grown at 37 ℃ in a humidified incubator with 5 % COMAP3K1 gene silencing was achieved by using a vector-based system to produce artificial miRNA. The amiRNA expression vector (pcDNA™6.2-GW/EmGFP-miR) was a gift from Dr. Sun HuaiChang, yangzhou university. We got the sequence of mice MAP3K1 gene from NCBI database (Accession number:NM_011945.2), with the help of INVITROGEN Block-iT RNAi Designer software, we analysed the MAP3K1 sequence and synthesis five candidate miRNA sequences with the highest star-rating (data not shown). Furthermore, Map3k1 amiRNA sequence was subcloned into the eGFP-GV273 vector and lentivirus was packed using the Lentivector Expression Systems .Total RNA was isolated from breast cancer cells by using Trizol (invitrogen), and cDNA was generated by reverse transcription system kit (invitrogen). Real-time PCR was used to detect relative gene expression using SYBR-Green real-time PCR mixes (Roche). GAPDH was used as the internal control, and the expression difference of MAP3K1 gene between every groups was analyzed by statistical software Graphpad prism 5.®800CW (ODYSSEY) for 2 h at room temperature. After additional washes, the protein bands were visualized by ODYSSEY System.Total protein was isolated from breast cancer cells using RIPA buffer containing 1 % PMSF, protein concentration was determined using a BCA protein assay kit . Protein samples were run in a 6 % seperating gel, and then transferred onto a nitrocellulose membrane. The membranes were blocked for 2 h at 4 ℃ with Tris-buffered saline-Tween 20 (TBST) containing 5 % skimmed milk, and probed with primary antibodies angainst MEKK1 (Santa Cruz), ERK and P-ERK , JNK and P-JNK (CST), P38 and p-p38 (CST), and internal control GAPDH (Santa Cruz). Antibodies were diluted in TBST containing 5 % skimmed milk. After 12 h incubation at 4 ℃, the membranes were washed in TBST 10 min for three times. Then, the membrane was incubated with Goat Anti-Rabbit IRDyeThe cells growing on the glass slide were fixed by 4 % paraformaldehyde, washed in PBS for 20 min, and then permeablized in 0.1 % Triton X-100/TBS for 10 min. Thereafter, the cells were blocked using 10 % goat serum for 1 h. After washing, slides were incubated with antibodies to MEKK1 overnight at 4 ℃. After washing, the cells were incubated with Cy3-conjugated anti-goat antibodies (Jackson ImmunoResearch) and 4,6-diamidino-2-phenylindole (DAPI) for 2 h. Images were captured using a Zeiss Axiovert microscope. EdU (Ribobio) and TUNEL (Promega) assays were performed according to manufacturers recommendations.Mammary and lung tissues were fixed in 4 % paraformaldehyde and embedded in paraffin for sectioning. Next, the sections were subjected to HE staining in accordance with the standard HE protocol. Breast cancer clinical pathologic specimens (provided by AFFINIATED HOSPITAL OF NANTONG NUNIVERSITY) were immunostained with an anti-MEKK1 antibody . Then, SP immunohistochemical staining kits was used according to manufacturers protocol (ZSGB-BIO).Wound-healing experiment was employed to detect the migration ability of 4T1 cells in vitro. 4T1 cells were transfected with Map3k1 amiRNA-3 or contol-amiRNA. When the cells reached 60 % confluence, 1 ml tip was used to scratch about 2 mm region along the plate Central line and the medium was replaced into serum-free medium. The cells were incubated at 37 ℃ for an additional 24 h. The images of cells were capture by Zeiss Axiovert microscope. The number of cells migrated to scratch region were analyzed to compare the migration ability of 4T1cells.5 cells were planted to invade through a filter (8 μm) toward 5 % serum-containing medium for 24 h. Thereafter, the invading cells were stained with Wright’s stian and counted for comparing the invasion ability of 4T1 cells.The invasion ability of 4T1 cells was assessed by Transwell (Corning) smeared with Matrigel (BD) assay. 4T1 cells transfected with Map3k1 amiRNA-3 or contol amiRNA were cultivated in serum-free medium, wherein 5 × 103 cells) was prepared in 0.35 % agar, which were added to a 6 cm plate over a base agar layer (0.6 %). Overlayed agar changed once a week, plates were incubated at 37 ℃ in a humidified incubator for 2 weeks. At termination, the colonies were stained with Crystal Violet and counted on a dissecting microscope. Positive colonies were defined as clusters consisting of more than 50 cells.A suspension of 4T1 cells (5 × 106 cells/mice) infected with control-amiRNA or Map3k1 amiRNA-3 lentivirus were injected into cleared fat pads of BALB/c-nude mice. Mice were examined for palpable tumors every 2 days, and the growth of the primary tumors was measured once a week using a vernier caliper. Two perpendicular diameters, termed length (L) and width (W), were determined, with length defined as the larger of the two measurements. Volume was calculated using the formula: 1/2 × l × W2, Mice were euthanized 4 weeks after injection of 4T1 cells. Primary tumors were removed and weighed. H&E staining was performed to determine the pathological structure of primary tumors.BALB/c-nude mice were employed to analyze the ability of tumorigenesis and lung metastasis of 4T1 cells in vivo. For assessing tumor incidence and growth, 4T1 cells (1.5 × 105 cells/mice) infected with control-amiRNA or Map3k1 amiRNA-3 lentivirus were transplanted into mice by tail vein injection. The weight of mice was measured once a week. Mice were euthanized 4 weeks after injection of 4T1 cells. Metastasis was evaluated in lungs, which is typical for this tumor due to their visibility and ease of quantification. Lungs were fixed in Bouin’s solution, and metastases were quantified by counting on a dissecting microscope. Finally, H&E staining was performed to analyze the pathological structure of lung metastasis of breast cancer.To assess the impact of MAP3K1-targeting miRNA on the metastasis of breast cancer, 4T1 cells . Values were compared using a student’s To explore the involvement of MEKK1 in breast cancer development, the expression pattern of MEKK1 was first determined using immunohistochemical analysis. As shown in Fig. Next, we investigated the role of MEKK1 in the regulation of breast cancer growth and invasion. Because recent studies highlighted that artificial miRNA could effectively target genes to modulate the physiology of cancer cells, we designed 5 MAP3K1-targeting miRNAs using Invitrogen Block-iT RNAi Designer program, and constructed the precursors of the 5 miRNAs into pcDNA™6.2-GW/EmGFP-miR vector. The constructs have been verified using PCR analysis and DNA sequencing (Data not shown). Thereafter, the constructs were transfected into 4T1 cells. Using real-time PCR analysis, we found that Map3k1 amiRNA-3 achieved the best interference efficiency . Next, the cells were subjected to EDU incorporation assay to determine the proliferation of the cells. As shown in Fig. Because ERK and p-38 were reported to play regulatory roles in the invasion of breast cancer cells, we addressed whether overexpression of Map3k1 amiRNA-3 might inhibit the migration and invasion of breast cancer cells (Gomes et al. Furthermore, we analyzed whether the apoptotic response were affected after Map3k1 amiRNA-3 overexpression. TUNEL assay was employed to determine the apoptotic rate of control and Map3k1 amiRNA-overexpressing 4T1 cells. As shown in Fig. To further delineate the involvement of artificial miRNA-induced MAP3K1 depletion in breast cancer progression, we investigated the in vivo growth and metastasis of breast cancer cells using nude mice model. Control and Map3k1 amiRNA-overexpressing 4T1 cells were injected into the flanks of BALB/c nude mice. The sizes of tumors were measured weekly, and the growth curves of the tumors were produced. As shown in Fig. To further elucidate the role of Map3k1 amiRNA in the regulation of breast cancer metastasis, we assessed whether Map3k1 amiRNA-3 could impair the ability of 4T1 cells to colonize the lungs of Balb/c nude mice. 4 weeks after cell injection, the mice were sacrificed and lung metastasis of breast cancer cells was examined. As shown in Fig. The MAPK cascades are central signaling pathways that play fundamental roles in many aspects of cellular processes, including proliferation, differentiation, migration and apoptosis. In the present study, we investigated the role and potential therapeutic value of MEKK1 in breast cancer. We found that MEKK1 was upregulated in a majority of breast cancer samples. Of great importance, we showed that miRNA-based MAP3K1 targeting significantly suppressed the proliferation and invasion of breast cancer cells. Additionally, using nude mice, we identified that Map3k1 amiRNA treatment attenuated the progression and lung metastasis of breast cancer in vivo. These data undoubtedly supported the notion that MEKK1 plays an important role in the regulation of breast cancer growth and metastasis, highlighting that miRNA-targeted MAP3K1 depletion is a valuable approach in the treatment of breast cancer.In recent years, the development of targeted therapy is a most attractive strategy of cancer therapy. In this regard, RNA-based modulation of mRNA and protein expression has attracted significant research attention (Tong et al. The oncogenic property of MEKK1 was largely attributed to the activations of downstream MAPK kinases. MAPK kinases play complicated roles in the development of human cancers. For example, ERK MAPKs are activated by a wide range of mitogens and play a predominant role in promoting the growth and metastasis of cancer, including breast cancer (Samatar and Poulikakos Taken all the experimental data in vitro and in vivo, we for the first time reported that MEKK1 was significantly upregulated in breast cancer specimens. Moreover, our findings supported that synthetic artificial miRNA that targets MAP3K1 exert remarkable growth-inhibitory and anti-metastatic effects in breast cancer cells, highlighting that artificial miRNA may confer significant anti-tumor activity through modulating the expression of oncoproteins.


In a phase III study of gemcitabine plus erlotinib for advanced pancreatic cancer conducted in Canada, the incidence of interstitial lung disease (ILD) was 3.5 %. However, the incidence of ILD was reported as high as 8.5 % in a Japanese phase II study. These results suggest the influence of ethnic factors in the association of the use of gemcitabine plus erlotinib with the incidence of ILD. Here, we conducted a prospective study to analyze the relationship between human leukocyte antigen (HLA) alleles and ILD in Japanese patients with advanced pancreatic cancer receiving gemcitabine plus erlotinib.2; administered by intravenous infusion on days 1, 8, and 15 every 4 weeks) and erlotinib . We compared the frequencies of HLA alleles in patients who did and did not develop ILD.Patients were treated with gemcitabine (1000 mg/mHLA-B*15:01 and DRB1*15:01 was observed in 2 of 4 patients (50 %) with ILD and in only 1 of 53 patients without ILD (2 %) resulting in odds ratio of 52.0 .A total of 57 patients were treated, and 4 patients (7.0 %) developed ILD. The combination of HLA-B*15:01 and DRB1*15:01 is associated with ILD in Japanese patients with advanced pancreatic cancer receiving gemcitabine plus erlotinib.These results suggest that the combination of The online version of this article (doi:10.1007/s00280-016-3026-6) contains supplementary material, which is available to authorized users. In a phase III study, gemcitabine plus erlotinib improved overall survival significantly compared with gemcitabine alone in patients with advanced pancreatic cancer . As sing>3 [hazard ratio (HR) 4.2 (95 % CI 2.2–8.2)] and concurrent/previous lung diseases [HR 2.2 (95 % CI 1.1–4.5)] [Risk factors for ILD identified in a post-marketing surveillance study of erlotinib plus gemcitabine (POLARIS) in Japanese pancreatic cancer patients were the number of metastatic sites at .1–4.5)] . In a ph.1–4.5)] .MUC) 5B gene as associated with familial interstitial pneumonia and idiopathic pulmonary fibrosis (IPF) in a white population [MUC4 gene and ILD induced by epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) or acute exacerbation of IPF was reported in Japanese patients [The factors described above do not explain the higher incidence of ILD in Japanese patients treated with gemcitabine, erlotinib, or their combination compared to that of white patients. However, genetic factors may explain the observed interethnic difference in ILD. A genome-wide linkage study identified the mucin  with histological or cytological evidence of unresectable locally advanced or metastatic pancreatic cancer were enrolled. Other eligibility criteria included an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0–1, adequate hematologic, renal, and hepatic functions, and a life expectancy of at least 2 months. Patients with a concurrent or previous history of ILD, IPF, pneumoconiosis, drug-induced pneumonia, pulmonary emphysema, or chronic obstructive pulmonary disease were excluded. Patients treated with radiation to the chest or lung resection, as well as gemcitabine or EGFR-TKI within 3 months, were also excluded.2 by intravenous infusion for 30 min on days 1, 8, and 15 every 4 weeks) and erlotinib  [Patients were treated with gemcitabine  and WAKFlow HLA typing kit . Data were analyzed using WAKFlow typing software  in the HLA Foundation Laboratory  .Chest X-ray was performed weekly for the first 4 weeks and every 2 weeks thereafter. Chest computed tomography (CT) scan was performed every 4 weeks. Antitumor efficacy was evaluated by CT every 8 weeks based on the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. Adverse events were assessed using Common Terminology Criteria for Adverse Events (CTCAE), version 4.0.P values <0.05 were considered statistically significant.Statistical analyses were performed using SPSS software version 21.0. Allele frequencies of HLA in patients with or without ILD were compared using Fisher’s exact test with 2 × 2 tables. We considered that at least 4 ILD events were necessary for the association study. In a Japanese phase II study of gemcitabine plus erlotinib for pancreatic cancer, the incidence of ILD was 8.5 %; therefore, the original planned sample size was approximately 50 to detect 4 ILD events.n = 44) and Kobe City Medical Center General Hospital (n = 13). Of these patients, 4 patients (7.0 %) developed ILD. Baseline characteristics are shown in Table Between February 2013 and July 2015, 57 patients were enrolled from two institutions: Kobe University Hospital (The median duration of the treatment was 1.7 months (range 1.0–3.3) in patients who developed ILD and 2.7 (0.1–16.0) months in those without ILD. The most common reason for discontinuation was disease progression  evaluated by RECIST criteria. Treatment-related adverse events lead to treatment discontinuation in 7 patients (12.3 %) due to malaise, diarrhea, dysgeusia, and ILD . Treatment was discontinued due to adverse events not related to the treatment in 4 patients (7.0 %). One patient (1.8 %) stopped the treatment due to progressive symptoms of the primary disease.Median progression-free survival was 2.8 (1.0–3.5) months in patients with ILD and 2.8 (0.1–16.8) months in patients without ILD. Partial responses were achieved in 2 patients (3.5 %), and stable disease (SD) was observed in 27 patients (47.4 %). Best response in four patients who developed ILD was SD in 1 and disease progression in 3 patients.Chest CT scans of 4 patients who developed ILD at the onset of ILD are shown in Fig. HLA-B*15:01, B*40:06, DRB1*09:01, and DRB1*15:01, but their association with ILD was not statistically significant. The frequencies of HLA alleles in patients with or without ILD are listed in the Supplementary Table. However, the combination of HLA-B*15:01 and DRB1*15:01 was observed in 2 of the 4 patients (50 %) who developed ILD, while only 1 of 53 patients (2 %) who did not develop ILD harbored this combination, resulting in an odds ratio (OR) of 52.0  of previously reported risk factors for ILD caused by gemcitabine and erlotinib [Our results demonstrate that the combination of rlotinib .-representation of HLA-DRB1*15:01 was reported in white patients with IPF not associated with drug therapy [HLA-A*3, B*14, B*15, or B*40, and combination of A2B15, A2B40, A11B15, A24B58, or A30B40) were also associated with IPF in a Han Chinese population [An over therapy . IPF is HLA-B*15:02 in Chinese patients and HLA-A*31:01 in Japanese or European patients [HLA-B*58:01 and HLA-B*57:01 are over-represented in allopurinol-induced severe cutaneous reactions [HLA-DQA1*02:01 or DRB1*07:01 [HLA alleles are associated with drug hypersensitivity. Carbamazepine-induced Stevens–Johnson syndrome is associated with patients –14. HLA-eactions , respectB1*07:01 . The freB1*07:01 , 19.HLA-A*31:01 and HLA-DRB1*15:02 are known to be associated with methotrexate-induced ILD in Japanese patients with rheumatoid arthritis (RA), which induce ILD as a complication [DRB1*15 and *16 were associated with a risk of ILD in Japanese RA patients regardless of methotrexate treatment [With regard to drug-induced ILD, lication , 21, andreatment .These observations suggest that HLA plays an important role in drug-induced ILD, and our findings demonstrate a significant association of certain HLA types with drug-induced ILD. The mechanism of the association between HLA and ILD is still unclear, although an in vitro study demonstrated that a direct interaction between HLA and carbamazepine activates T cells . Drugs oHLA-B*15:01/DRB1*15:01 was observed. Differences in the population frequency of the HLA alleles may explain, at least in part, the interethnic differences in the incidence of ILD induced by gemcitabine and erlotinib.The incidence of ILD by gemcitabine plus erlotinib for advanced pancreatic cancer was 8.5 % in a Japanese phase II study, 6.2 % in a post-marketing surveillance study, and 7.0 % in our study. We carefully excluded patients with risk factors for ILD from our study. Nonetheless, a significant number of patients developed ILD. These incidences are obviously higher than those reported in white patients. In our prospective study, an association between drug-induced ILD and HLA-B*15:01 and DRB1*15:01 is associated with ILD in Japanese patients with advanced pancreatic cancer receiving gemcitabine plus erlotinib.In conclusion, these results suggest that the combination of Supplementary material 1 (DOC 103 kb)Below is the link to the electronic supplementary material.