We also demonstrate that blockade of Syntaxin 1 abolishes neurotrophin-dependent growth of axons in neuronal cultures and decreases exocytotic events at the tip of axonal growth cones. 25-kDa soluble N-ethylmaleimide-sensitive factor attachment protein and Vesicle-associated membrane protein 2 do not participate in the formation of this SNARE complex, while tetanus neurotoxin-insensitive vesicle-associated membrane protein interacts with Trk receptors; knockdown of this (v) SNARE impairs Trk-dependent outgrowth. Taken together, our results support the notion that an atypical SNARE complex comprising Syntaxin 1 and tetanus neurotoxin-insensitive vesicle-associated membrane protein is required for axonal neurotrophin function.SNARE proteins are essential components of the machinery that regulates vesicle trafficking and exocytosis. Their role is critical for the membrane-fusion processes that occur during neurotransmitter release. However, research in the last decade has also unraveled the relevance of these proteins in membrane expansion and cytoskeletal rearrangements during developmental processes such as neuronal migration and growth cone extension and attraction. Neurotrophins are neurotrophic factors that are required for many cellular functions throughout the brain, including neurite outgrowth and guidance, synaptic formation, and plasticity. Here we show that neurotrophin Trk receptors form a specific protein complex with the t-SNARE protein Syntaxin 1, both  It has  turning  or the c turning , dependil stores , 22.Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) are members of the neurotrophin family of growth factors. They are all polypeptides that support the growth, differentiation and survival of neurons in the developing nervous system and maintain neurons in the mature nervous system –25. NeurIndeed, neurotrophins play an important role in regulating various processes, including neural survival, development, function, and plasticity –30. ManyMoreover, the original discovery that Trks are oncogenes has led to the association of their expression with the biology of tumors; their expression and signaling cascades are often modified not only in tumors of neural origin but also in carcinomas, myelomas, and prostate and lymphoid tumors , 36.in vivo [In previous studies, we demonstrated that the t-SNARE protein Sytx1 is involved in DCC/Netrin-1-dependent chemoattraction , 18 and in vivo . Those fSNARE protein complexes are also overexpressed in several cancer subtypes, thereby once again suggesting a strong link between cancer and neural development. In particular, HER2-enriched breast tumors overexpress Sytx1A . AdditioHere we addressed whether Trk-mediated neurotrophin effects on neurite outgrowth require the involvement of SNARE proteins. We show that Trk receptors interact with Sytx1 and TI-VAMP and that these SNARE proteins are necessary for neurotrophins to induce neurite outgrowth via exocytosis.TM-JX-TK-Y817 (tagged with myc) and Sytx1AEGFP. In lysates immunoprecipitated with anti-myc antibodies, Sytx1AEGFP was detected by WB using anti-GFP. Pull-downs with anti-GFP antibodies revealed TrkB protein. No co-immunoprecipitation was detected when cells were transfected with Sytx1AEGFP or TrkBTM-JX-TK-Y817 DNA alone . In lysates immunoprecipitated with anti-HA antibodies, Sytx1A-CMV was detected by WB using anti-HPC1. Pull-downs with anti-HPC1 antibodies revealed TrkA protein  Figure , and VAMTo confirm these findings, a series of co-immunoprecipitation analyses were also performed in lysates from embryonic (E15) and adult brains. We did not detect co-immunoprecipitation of TrkB with either SNAP25 or VAMP2. This observation was then confirmed by immunoprecipitating VAMP2 and SNAP25 and immunoblotting with anti-TrkB .As the formation of the SNARE complex involves v-SNAREs and t-SNAREs, we next studied the possible interaction between the t-SNARE TI-VAMP and the Trk receptor. We co-immunoprecipitated HEK293 cells cotransfected with DNAs encoding for TrkB-GFP and TI-VAMP. In lysates immunoprecipitated with anti-GFP, TI-VAMP was identified by WB using anti-TI-VAMP Figure . The revAdditionally, we treated hippocampal primary cultures with BDNF for 0, 15 and 30 min and analyzed the co-localization between TrkB and TI-VAMP. Confocal images and intensity correlation analysis indicated an increase in the co-localization of both proteins with respect to the control Figure . TogetheTK); the transmembrane-juxtamembrane-tyrosine kinase domain, carrying the analogous tyrosine 490 (Y490) in TrkB receptors (TrkBTM-JX-TK); the juxtamembrane-tyrosine kinase domain, lacking the transmembrane domain (TrkBJX-TK-Y817) but including the Y817 analogous to the Y785 of TrkA receptors; and the entire intracellular domain, which carries all the phosphorylatable catalytic tyrosine residues, which included the transmembrane domain (TrkBTM-JX-TK-Y817)  Figure . DNAs we) Figure . TrkB DNH3), transmembrane (Sytx1ATM), H3-transmembrane (Sytx1AH3TM), and the Habc (Sytx1AHabc) domains, as well as the complete cytosolic tail (Sytx1ACYT) and a full-length construct (Sytx1AFL) . We used six constructs carrying the H3 (Sytx1A) Figure . The pre) Figure . Constru) Figure . Co-tranThe sensory neurons of the Nodose ganglia are a classic example of peripheral nervous system neurons that do not require NGF for survival or outgrowth during development. However, they are dependent on BDNF and to a lesser extent on NT-3 and NT-4 , 44. In Nodose Figure  and DRG To corroborate these data, we next used dissociated primary Nodose cultures supplemented with BDNF and treated them with BoNT/C1, BoNT/A and TeNT Figure . IndividH3TM construct containing the minimal Sytx1A interaction domain, as a dominant negative to interfere with Sytx1 . We cloned Sytx1AH3TM into a pIRES-EGFP vector (Clontech) to generate a unique transcript capable of translating both target and EGFP proteins. We also took advantage of two shRNAs for TI-VAMP (D06 and D07) and two shRNAs for Sytx1A (shRNA1 and shRNA2). The shRNA sequences were tested in transfected cells to assess the percentage of silencing. D06 TI-VAMP shRNA induced a 50% decrease in expression while D07 TI-VAMP shRNA achieved about 85% . Together, these findings indicate that interference with Sytx1 function via expression of a Sytx1AH3TM dominant negative or using siRNAs blocks BDNF-mediated axonal growth in dissociated neurons  and red channels (exocytosis vesicles). The results of these pulse–chase experiments revealed an initial accumulation of Bodipy-ceramide label (red channel) in the Golgi apparatus, followed by the sequential appearance of vesicle-like structures in the cell body, neurites, and growth cones Figure . We quanIn order to corroborate that VAMP2 and SNAP25 were not involved in this mechanism, we stimulated Nodose neurons with BDNF and simultaneously treated them with BoNT/A and TeNT. As in BDNF treatment, fluorescence decreased at 15 and 30 min, thereby demonstrating the fusion of exocytotic vesicles at the membrane Figure .These results indicate that Sytx1 cleavage abolishes the exocytosis of pre-labeled vesicles and demonstrate that Sytx1 is required for BDNF-regulated exocytosis in the growth cone.Axonal growth is a complex event in which the mechanisms that regulate elongation, fasciculation and guidance are closely coordinated. During pathfinding, growth cones exhibit a variety of behaviors, such as cone collapse, repulsion and attraction , 53–57. in vivo and in vitro with Sytx1, but not with VAMP2 or SNAP25. Furthermore, co-immunoprecipitation assays in vitro revealed that TrkA and TrkC also interact with Sytx1, thereby supporting the notion of a common relationship between Trk receptors and the proteins regulating exocytosis. The interaction of Trk receptors with Sytx1 was confirmed by co-immunoprecipitation assays using specific domains of these proteins. Our results also suggest that this interaction is regulated by Trk ligands, since BDNF treatment of hippocampal growth cones increased TrkB/Sytx1 co-localization. Thus, together with our previous findings on the Netrin-1 receptor [The addition of new membrane to nerve processes is an essential step in nerve growth and differentiation and also in axonal guidance , 66, 67.receptor , 18 and receptor , 68, 69,receptor . Howeverreceptor . In addireceptor . With rereceptor . Similarreceptor . VAMP2 mreceptor .Exocytosis requires at least one v-SNARE protein, in addition to Sytx1. In previous work, we found that DCC receptors interact with TI-VAMP in a Netrin-dependent manner to induce exocytosis in growth cones . TherefoThe previous results point to a biochemical and functional link between the receptors for trophic factors and the proteins that regulate exocytosis. We demonstrate that treatment with BoNT/C1 reduces axonal elongation in Nodose and DRG explants, as a result of the cleavage of Sytx1. Finally, we sought to address how SNARE proteins allow the elongation of neuronal axons by an exocytotic process. To this end, we used Bodipy-ceramide to explore exocytosis induction during BDNF-dependent axonal growth in Nodose explants. Incubation with BDNF led to the release of pre-labeled vesicles at the cone tips Figure . AdditioOur results might also explain why Sytx1 is an absolute requirement for neuronal survival and maintenance . Recent In conclusion, our results demonstrate that neurotrophin/Trk receptor signaling requires two SNARE proteins, namely Sytx1 and TI-VAMP, to mediate axonal outgrowth and extension. Together with previous studies showing that various SNARE complex proteins are necessary for the signaling of guidance molecules exerting either chemoattraction  or OF1 embryos and adult mice  were used. The mating day was considered embryonic day 0. Pregnant females at the development stage of interest were killed by cervical dislocation, following the European Community Council directive and the National Institutes of Health guidelines for the care and use of laboratory animals. Experiments were also approved by the local ethics committees.7 cells in 90-mm plates for biochemical analysis. Cells were cultured for 3 days in Neurobasal medium containing 1% glutamax and 2% B27 supplement. Primary cultures were incubated with BDNF for 0, 15, and 30 min .E16 mouse brains were dissected out to obtain hippocampal neuronal cultures in PBS containing 0.6% glucose. The brains were then dissociated by incubation for 12 min at 37°C with 0.05% trypsin (Invitrogen) and treated for 10 min with DNase (Roche Diagnostics); tissue pieces were dissociated by gentle sweeping. Cells were then counted and seeded at a density of 50,000 neurons per dish in 4-well plates on coverslips pre-coated with poly-D lysine (Sigma) for immunocytochemistry and at a density of 3 million neurons per well in 6-well plates and 10For Nodose primary cultures, E14 mouse embryos were dissected in F12 media (Gibco-BRL). Ganglia were dissociated for 10 min at 37°C with 0.05% trypsin (Invitrogen), and the tissue pieces were dissociated by gentle sweeping. Cells were then counted and seeded at a density of 5,000 neurons per dish in 4-well plates on coverslips pre-coated with polyornithine  and laminin (20 ug/ml for 2 h) for immunocytochemistry. The neurons were incubated for 24 h in a defined media containing Dulbecco’s Modified Eagle’s Medium , 10% Fetal Bovine Serum (FBS), 0.54% glucose, 1% glutamine, 1% Penicillin/ streptomycin, N2 and B27, and supplemented with 50 ng/ml BDNF (Promega). The day after, primary cultures were deprived for 2 h and then treated with 100 ng BDNF or control medium for 0, 5, 15 and 30 min.Hippocampal explants were obtained from E15 brains, sectioned using a tissue chopper. Small tissue explants (300–400 µm thick) were obtained from the CA1/CA3 regions . HippocaNodose explants were incubated on day 2 and day 3 respectively, with the Bodipy-labeled-ceramide–BSA complex (Invitrogen). To allow internalization and accumulation of the labeled lipid complex in the Golgi apparatus, explants were incubated for 30 min at 37°C in their own medium and washed 5 times. To allow transport to the cell surface, they were further incubated at 37°C for 2 or 3 h , 78. TheQuantitative analysis of the confocal images of growth cones was performed by measuring the average pixel intensity of each image with an ImageJ macro. The macro transformed all the images (red and green) into a 32-bit depth and then ran a threshold between 10 to 255 gray levels to avoid background from 0 to 10 gray levels. Finally, we measured the area and the mean gray value for each channel. The average pixel intensity was then calculated from this value on the basis of the ratio between red and green channels. The values were classified in function of the treatment time. At least 5 separate experiments  were carried out for each condition. The number of axonal growth cones counted is shown in the figure legends.TM-JX-TK-Y817); pCMVtag3a-TM-JX-TK-TrkB (TrkBTM-JX-TK); pCMVtag3a-JX-TK-TrkB (TrkBJX-TK-Y817); and pCMVtag3a-TK-TrkB (TrkBTK).HEK293 cells were cultured in DMEM, 10% FBS, 0.5% glutamine and 1% penicillin/ streptomycin. All cells were maintained in 5% CO2 at 37°C. For transfection, cells were cultured overnight to 60–70% confluence in 90-mm culture plates. The transfection was performed in Opti-MEM medium with Lipofectamine Reagent Plus, following the manufacturer’s instructions . Cells were transfected with different combinations of cDNAs: pEF-BOS-SNAP25-FLAG and pEF-BOS-VAMP2-FLAG ; TI-VAMP ; pcDNA-TrkA-HA (kindly provided by Dr. D. Martin-Zanca); pEGFPc1-TrkB ; and pcDNA-TrkC-myc ; pCMVtag3a-TM-JX-TK-Y817-TrkB (TrkBTM-JX-TK-Y817 fragment was obtained by PCR amplification of pEGFPc1-TrkB using the following primers: Forward 5′TATATGAATTCTATGCATCTCTCGGTCTATGCT3′ and Backward 5′ATATATAAGCTTCTAGCCTAGGATGTCCAGGTA3′). The TrkBTM-JX-TK fragment was obtained by PCR amplification of pEGFPc1-TrkB using the following primers: Forward 5′TATATGAATTCTATGCATCTCTCGGTCTATGCT3′ and Backward 5′ATATATAAGCTTCTACGCCTTCGCCAAGTTCTG3′. The TrkBJX-TK-y817 fragment was obtained by PCR amplification of pEGFPc1-TrkB using the following primers: Forward 5′TATATGAATTCTATGAAGTTGGCGAGACATTCC3′ and Backward 5′ATATATAAGCTTCTAGCCTAGGATGTCCAGGTA3′. Finally, the TrkBTK fragment was obtained by PCR amplification of pEGFPc1-TrkB using the following primers: Forward 5′TATATGAATTCTATGCACAACATCGTTCTGAAG3′ and Backward 5′ATATATAAGCTTCTACGCCTTCGCCAAGTTCTG3′.The TrkB truncated fragments were obtained by PCR amplification of pEGFPc1-TrkB using specific primers and subsequently cloned into EcoRI/HindIII sites in the pCMVtag3a expression vector (Clontech). The TrkBFull-length Sytx1A or truncated DNAs  were cloned into EcoRI sites in pEGFPC1 fusion mammalian protein expression vector (Clontech), as described .Using the NeonTM transfection system (Invitrogen), nodose-dissociated primary cultures were transfected with one of the following DNAs: pEGFPC1, Sytx1AH3TMEGFP, shRNA TI-VAMP D06  (TI-VAMP shRNAD06) and shRNA TI-VAMP D07  (TI-VAMP shRNAD07). For Sytx1 RNAi experiments, we used a pSUPER.retro.puro plasmid  and specific oligonucleotides of the Sytx1A sequence: 5′GATCCCCCCAAGAAGGCCGTCAAGTATTCAAGAGATACTTGACGGCCTTCTTGGTTTTT3′ (Forward), and 5′AGCTAAAAACCAAGAAGGCCGTCAAGTATCTCTTGAATACTTGACGGCCTTCTTGGGGG3′ (Reverse)  .Embryonic and adult brains, as well as transiently transfected HEK293 cells and primary cultures, were collected in hypotonic lysis buffer  containing a protease inhibitor cocktail (Roche). Thereafter, cells were centrifuged at 13,000 rpm for 20 min, and the pellet was discarded. The supernatant was analyzed by Western blot or subjected to immunoprecipitation. For tissue extracts, samples were weighed and 3 volumes of lysis buffer was added. The mixture was processed using a Polytron homogenizer, and insoluble material was removed by centrifugation at 13,000rpm for 20 min.Protein concentration was determined by means of the Bradford assay. For the immunoprecipitation assays, 200–400 µg of total protein per sample was used. Brain lysate homogenates were incubated overnight at 4°C with anti-TrkB , anti-Sytx1 , anti-SNAP25 , or anti-VAMP2  in IP buffer . Transfected HEK293 cell homogenates were incubated with anti-FLAG , anti-myc , anti-HA , anti-GFP , or anti-TI-VAMP (1:200 Abcam) antibodies. All the assays were performed using Protein G-sepharose beads (Sigma) for 2 h at 4°C. After three washes, 1 volume of 3x Lamelli loading buffer /SDS-sample buffer was added to the beads, and the proteins were analyzed by SDS-PAGE and Western blot. Samples were run in polyacrylamide gel at the opportune concentration at 120V. Afterwards, proteins were transferred onto nitrocellulose membranes overnight at 135 mA at 4°C. The membranes were blocked with 5% non-fat dry milk in Tris-HCl-buffered saline (TBS) containing 0.1% Tween 20, and incubated overnight at 4°C with anti-TrkB , anti-Sytx1 , anti-SNAP25 (SMI-81 clone 1:500 Sternberger Meyer), anti-VAMP2 , mouse anti-TI-VAMP (1:200 Abcam), rabbit anti-GFP , mouse anti-FLAG , mouse anti-HA , or mouse anti-myc  antibodies. After incubation with secondary antibodies, blots were developed following the ECL method (Amersham Pharmacia Biotech).To normalize the data, we represented the ratios between the amount of co-immunoprecipitated protein  and the immunoprecipitating protein (Sytx1) (expressed as percentage association with respect to controls).Nodose explants, DRG explants, and primary cultures were fixed with paraformaldehyde 4% PBS 0.1M (pH 7.4) for 30 min and washed with PBS 0.1M several times. The cultures were then permeabilized and blocked for 3 h with PBS, Normal Goat Serum 2% and Triton X-100 0.1% (Sigma). Finally, they were incubated overnight at 4°C with primary antibody against neuron-specific class βIIITubulin , rabbit anti-GFP , mouse anti-Sytx1 (1:500), rabbit anti-TrkB , or TI-VAMP , followed by incubation with Alexa-fluor 488- and Alexa-fluor 568-coupled corresponding secondary antibodies (1:500) and DAPI (Sigma) for 2 h at room temperature. The coverslips were then washed and mounted on glass slides with Mowiol.The images were captured by an E1000 fluorescence microscope from Nikon, using a 4X0.13NA and 10X 0.30 NA lens. Metamorph software and a CoolSnap fix scientific CCD camera were used for acquisition. Explants treated with toxins were quantified using Image J by calculating the area in pixels occupied by the axons. A background pixel level was defined and used to subtract the former value, and upper and lower density limits were defined for each explant. Once the segmentation range was defined, any pixels falling within this range were automatically counted. To quantify the area occupied by neurites emanating from explants, the tissue explant was manually drawn and excluded from the picture, thereby giving an area measurement for the pixels occupied by the neurites alone. The values obtained were plotted on a bar chart with their standard deviation. Data were collected from at least three independent experiments (3–7 experiments per condition). The number of cultures quantified is shown in the Figures.Confocal images of axonal growth cones  were taken with a Leica TCS SPE  confocal scanning microscope adapted to an upright LEICA DM 2500 microscope. Samples were scanned using 40x 1.15 NA and 63x 1.3NA oil objectives. We used a zoom ranging from 1 to 3.5 to analyze intracellular regions and the LAS AF software from Leica.Fluorescent images were processed using open software Image J. PDM images ((red intensity-mean red intensity)×(green intensity-mean green intensity)) were calculated and normalized for each pixel of an image and expressed as the degree of co-localization of two signals, with a value varying from –1 to +1, where –1 indicates maximal exclusion, negative values asynchronous localization, positive values synchronous localization , and +1 maximal co-localization. PDM images and Mander’s overlap coefficients were calculated using ImageJ “Intensity Correlation Analysis plug-in” .The entire images of coverslips were detected by an automated inverted fluorescence wide-field microscope with high content screening ScanR from Olympus IX81 using a 20x objective lens and a Hamamatsu Orca-ER digital camera . All the captured images were used to run an ImageJ plug-in “Stitch grid of images” and obtain a single mosaic image for each coverslip. The total image of the coverslips was used to calculate the total length of each axon, while the Neuron J plug-in was used to calculate ramification. The total length of each experimental group was normalized to control conditions.Data were expressed as means ± SEM and were analyzed using the Kruskal-Wallis test, followed by the Bonferroni Post-hoc test (Statgraphics Centurion XVII.I).The datasets generated or analyzed during the current study are available from the corresponding authors on request.


Little has been known about the role of non-coding RNA regulatory network in the patterns of growth and invasiveness of gastric cancer (GC) development.MicroRNAs (miRNAs) microarray was used to screen differential miRNA expression profiles in Ming’s classification. The significant differential expressions of representative miRNAs and their interacting circular RNA (circRNA) were confirmed in GC cell line and 63 pairs of GC samples. Then, a circRNA/miRNA network was constructed by bioinformatics approaches to identify molecular pathways. Finally, we explored the clinical value of the common targets in the pathway by using receiver operating characteristic curve and survival analysis.Significantly differential expressed miRNAs were found in two pathological types of GC. Both of miR-124 and miR-29b were consistently down-regulated in GC. CircHIPK3 could play a negative regulatory role on miR-124/miR-29b expression and associated with T stage and Ming’s classification in GC. The bioinformatics analyses showed that targets expression of circHIPK3-miR-124/miR-29b axes in cancer-related pathways was able to predict the status of GC and associated with individual survival time.The targets of circHIPK3-miR-124/miR-29b axes involved in the progression of GC. CircHIPK3 could take part in the proliferation process of GC cell and may be potential biomarker in histological classification of GC.The online version of this article (10.1186/s12967-018-1582-8) contains supplementary material, which is available to authorized users. Gastric cancer (GC) is an important public health problem in most parts of the world –3. AlthoGC tissue is composed of cancer cells and various types of stromal cells including endothelial cells and fibroblasts. Cell proliferation and invasion are the main pathological features of GC, which drive malignant tumor cells transfer from a primary site to other areas . Based oMicroRNA (miRNA) play critical biological roles in human carcinogenesis through various molecular mechanisms. Using miRNA-profiling-based screening assay can distinguish types and stage of cancers, and some specific miRNAs may be associated with certain histological subtypes of cancer –14. CircThe snap-frozen GC tissues and matched normal gastric epithelial tissues were recruited from the patients receiving operation in Zhongshan Hospital, Xiamen University. All surgical specimens were confirmed by pathological examination. Tumors were staged according to the tumor-node metastasis (TNM) staging system (7th ed.). Histological grade was on the basis of the National Comprehensive Cancer Network (NCCN) Clinical Practice Guideline of Oncology (V.1.2012). A total of 63 pairs of clinical samples were included, 28 infiltrative type GC samples and 35 expanding type GC samples. No chemotherapy or other antitumor treatments were received at the time when the specimens were obtained.2.The XGC-1 cell line originated from infiltrative type GC and the XGC-2 cell line was established from expanding type GC. The human gastric epithelial cell line GES-1 was obtained from the Cancer Institute and Hospital of the Chinese Academy of Medical Sciences . Two GC cell lines, MGC-803 and BGC-823, were purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences . All the cells were cultured in RPMI1640 medium  with 10% fetal calf serum and incubated at 37 °C in a humidifed atmosphere with 5% COThe RNA extraction was performed as previous report . Three pAll real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) process were performed as previous report , 28. TheTo investigate the biological effect of circHIPK3 in GC cells, circHIPK3 over-expression vector was constructed by using the pHB-circBasic™ circular RNA cloning kit . The specially designed front cir-signal and back cir-signal were synthesized and added to the downstream of the CMV promoter in the pHB-circBasic™ vector. In brief, the cDNA encoding liner form of HIPK3 transcript in GES-1 cells was amplified using primers 5-GTATGGCCTCACAAGTCTTG-3 and 5-CTGTAGTACCGAGATTGTAGATATG-3, and then the PCR product was purified by using Gel Extraction Kit . Firstly, we amplified the purified PCR product using the circular primers 5′-CGTACTAATGACTTTTTTTTTATACTTCAGGTATGGCCTCACAAGTCTTG-3′ and 5′-CCTAATTCTTTTCCTTGCTTCTTACCTGTAGTACCGAGATTGTAGATATG-3′. Subsequently, this PCR fragment was purified again and inserted into the site between the front cir-signal frame and back cir-signal frame in the pHB-circBasic™ vector. The result of this fusion vector construction was verified by Sanger sequencing. Finally, GC cells were transfected with circHIPK3 plasmids using the Turbofect transfection reagent . The siRNA was transfected into cells using ribo FECT ™ CP Transfection Kit . The proliferation of GC cell, MGC-803 and BGC-823, were tested by Cell Counting Kit-8 (CCK-8) assay  and performed as previous report .ONCOMINE gene expression array datasets was used to explore gene expression profiles in human cancer. In this study, we analyzed the datasets of Cui , which rP value as previous report [Overall survival (OS) and first progression (FP) curves were calculated with the Kaplan–Meier method to evaluate the prognostic value of COL1A1, COL4A1 and CDK6 mRNA expression in GC. A total of 876 GC patients were recruited from the Kaplan–Meier Plotter online database. Subjects were split into two groups by median expression (high vs. low expression) and assessed by a Kaplan–Meier survival plot, with the hazard ratio (HR) with 95% confidence intervals (CI) and logrank s report , 33.t test or one-way ANOVA. Spearman′s rank test was used to analysis the correlation between miRNAs expression and circHIPK3 levels. P < 0.05 was considered statistically significant. All statistical analysis was performed using Statistical Program for Social Sciences (SPSS) software 17.0 .Data between experimental groups were analyzed by the Student′s P < 0.05). In the infiltrative GC, 13 miRNAs were detected to be differentially regulated, among which 4 miRNAs were up-regulated, while 9 miRNAs were down-regulated compared to their paired normal tissue. 27 miRNAs were differentially expressed in expanding type GC, among which 9 miRNAs were up-regulated and 18 miRNAs down-regulated compared to their paired normal tissue, respectively . In addition, the expression of some miRNAs associated with Ming’s classification were further verified in Ming’s classification related GC cell lines (XGC-1 and XGC-2), GES-1 was used as control  of these pathways were compared. Among the 8 pathways, the −log2 scaled by P-value of the ECM-receptor interaction showed an infinite value  could be regulated by both miR-124 and miR-29b in the network, suggesting that they might act crucial roles mediated by circHIPK3-miR-124/miR-29b axes. Then, we used Oncomine cancer microarray mRNA database to indentify the expression levels of these three genes in GC samples. All of these genes showed significantly higher expression in 80 paired cases study .We then ask if the mRNA levels of these target genes mediated by circHIPK3-miR-124/miR-29b axes were down or up-regulated in GC. As shown Figs. a, b, thrP < 0.001; AUC = 0.775, P < 0.001, respectively) but not CDK6 expression .Furthermore, the clinical value of COL1A1, COL4A1 and CDK6 were also assessed. ROC curve showed that the status of GC was able to be predicted by using expression levels of COL1A1 and COL4A1  and FP  than those with the lower levels of COL1A1. Similar result was also found in survival analysis about mRNA expression of COL4A1  and CDK6 .As the target genes showed aberrantly expression profiles, we asked whether the target genes could serve as prognostic markers in GC patients. Therefore, overall survival (OS) and first progression (FP) curves were plotted by using Kaplan–Meier method according to the gene expression level in 876 GC samples. Patients with higher levels of COL1A1 had a significantly shorter OS  Statistically significant correlations were revealed between miR-29b or miR-124 and their mediated pathways by P-value  in heatmap. Red represents high significance. (B) The relevant pathways of miR-29b mediated, miR-124 mediated, and miR-29b and miR-124 jointly mediated. (C) The magnitudes of the significant correlation about Pathways among miR-29b mediated, miR-124 mediated, and miR-29b and miR-124 jointly mediated by P-value .Additional file 3: Fig. S2. Mapping of Pathways in cancer mediated by miR-124 and miR-29b. Yellow marked nodes are associated with target genes regulated by circHIPK3-miR-124/miR-29b axes enrichment on this pathway and devoted to the initiation and progression of GC.Additional file 4: Table S2. Detail information of enriched clusters.Additional file 5: Fig. S3. Effect of circHIPK3 on cell proliferation.  Knockdown of circHIPK3 inhibits human GC cell proliferation.  Over-expression of circHIPK3 promoted the cell proliferation. NC, negative control. *P < 0.05, **P < 0.01, ***P < 0.001.


Therefore, a selective and sensitive fluorescent method was developed. It has a linear response of 0.1–2.0 and 2.0–25.0 μM and a detection limit of 0.067 μM (S/N = 3). The fluorescent sensing platform was also used to detect L-carnitine in human serum and milk samples, which provided potential applications for the detection of drugs abuse and had path for guarding a serious food safety issues.Supramolecular host-guest interaction and sensing between cationic pillar[5]arenes (CP5) and L-carnitine were developed by the competitive host-guest recognition for the first time. The fluorescence sensing platform was constructed by CP5 functionalized Au nanoparticles (CP5@Au-NPs) as receptor and probe , which shown high sensitivity and selectivity for L-carnitine detection. Due to the negative charge and molecular size properties of L-carnitine, it can be highly captured by the CP5 via electrostatic interactions and hydrophobic interactions. The host-guest mechanism between PP5 and L-carnitine was studied by  Gold nanoparticles (Au-NPs) have the characteristics of facile synthesis, high chemical stability and easy surface functionalization, which have attracted the great interest of researchers in the fields of nanotechnology and nanomaterials because of their importance in biomedicine  sensing para-positions to form a unique rigid pillar architecture. Pillar[n]arenes are important players in supramolecular chemistry due to their easy synthesis, unique pillar shape, symmetrical structure, excellent host-guest properties and natural supramolecular assembly characteristics arenes (CP5) and L-carnitine. The fluorescence sensing platform is constructed by CP5@Au-NPs as receptor and probe R123, which has high sensitivity and selectivity to detect L-carnitine. A competitive fluorescence sensing platform based on CP5@Au-NPs is illustrated in 4, Rhodamine 123 (R123), L-carnitine and NaBH4 were obtained from Shanghai Titan Scientific Co. Ltd . CP5 was synthesized by the literature arene-modified Au nanoparticles were prepared.The CP5@Au-NPs composite was obtained based on similar work ,34. The −1) were prepared. A final concentration of 2 μM R123 was also obtained via dilution. By gradually addition of CP5@Au-NPs dispersion to the R123 solution and the fluorescence of the R123 was gradually quenched. The competitive displacement experiments were performed as follows: the L-carnitine solution was gradually added to a complex of R123-bound CP5@Au-NPs to displace the R123 molecule from the cavity of CP5 by L-carnitine. The fluorescence signal was measured and recorded after the combined solution was mixed by vortexing for 3 min.Aqueous solutions of R123 (200 μM), L-carnitine (400 μM) and CP5@Au-NPs (1.0 mg mL4] at different concentrations was obtained according to the surface plasmon resonance (SPR) of Au-NPs at ~520 nm, as shown in 4] was increased from 0.05 to 1, which suggested that the excess CP5 had little influence on the sizes of Au-NPs. The synthesized CP5@Au-NPs was wine red  and the corresponding regression equations of F/F0 = 0.41 C (µM) + 1.13 and F/F0 = 0.07 C (µM) + 1.48 with correlation coefficients of 0.925 and 0.995 were obtained. This approach was compared to other methods for detection L-carnitine (arnitine . This co3)3+ in CP5  and the structures of these interferences were shown in In summary, we describe a simple and convenient “turn-off-on” fluorescent sensing platform that uses cationic water-soluble pillar[5]arene modified Au-NPs and dye Rhodamine 123 as energy donor-acceptor pair. The outstanding host–guest recognition capability of CP5 and the excellent quenching performance of Au-NPs make this sensing system suitable for L-carnitine detection in human serum and milk samples. This work demonstrates that the CP5@Au-NPs composite is a good energy acceptor for fluorescence sensing platforms with potential applications in many fields.


Citrobacter rodentium is a gastrointestinal infection that requires early IL-22 from group 3 innate lymphoid cells (ILC3) for resistance. The role of vitamin D in the clearance of C. rodentium infection was tested in vitamin D sufficient (D+) and vitamin D deficient (D-) wildtype (WT) and Cyp27B1 (Cyp) KO mice 2D, 1,25D). Feeding Cyp KO mice D- diets reduced vitamin D levels and prevented synthesis of 1,25D. D- (WT and Cyp KO) mice had fewer ILC3 cells and less IL-22 than D+ mice. D- Cyp KO mice developed a severe infection that resulted in the lethality of the mice by d14 post-infection. T and B cell deficient D- Rag KO mice also developed a severe and lethal infection with C. rodentium compared to D+ Rag KO mice. D- WT mice survived the infection but took significantly longer to clear the C. rodentium infection than D+ WT or D+ Cyp KO mice. Treating infected D- Cyp KO mice with IL-22 protected the mice from lethality. Treating the D- WT mice with 1,25D reconstituted the ILC3 cells in the colon and protected the mice from C. rodentium. IL-22 treatment of D- WT mice eliminated the need for vitamin D to clear the C. rodentium infection. Vitamin D is required for early IL-22 production from ILC3 cells and protection from enteric infection with C. rodentium. Cyp27B1e system . Restinge system , 9. Takein vitro and in vivo have been suggested as mechanisms underlying the ability of 1,25D to suppress IBD  mice  ress IBD –16. Becanfection . ConversWT) mice , 20. 1,2C. rodentium is a gram-negative murine bacterial pathogen, and a model of human infection with enterohemorrhagic and enteropathogenic Escherichia coli infections. WT mice clear C. rodentium infection within 4–5 weeks of infection  were used to model severe vitamin D deficiency D  WT and D+ Cyp KO mice. There was no difference in the susceptibility of D+ WT and D+ Cyp KO mice to C. rodentium. D- mice had reduced numbers of ILC3 cells that produced IL-22 in the colon compared to D+ mice. In addition, the expansion of colonic Th17 cells that make IL-17 and IL-22 were lower following C. rodentium infection in D- mice. 1,25D treatment of D- mice recovered ILC3 cells and increased the rate of C. rodentium clearance compared to D- mice. Treatment of D- Cyp KO mice with IL-22 prevented the lethality and treatment of D- WT mice with IL-22 eliminated the need for vitamin D in the clearance of C. rodentium. Vitamin D is required for ILC3 derived IL-22 in the colon and the induction of protective Th17 cell expansion following infection. In the absence of vitamin D, mice develop a more severe enteric infection that takes longer to resolve.Here the effect of vitamin D status on mucosal immunity to tm2Litt/J (Rorcgfp) mice were originally from the Jackson Laboratories  and bred for experiments at the Pennsylvania State University . C57BL/6 Cyp27B1 (henceforth described as Cyp KO) KO breeders were a gift from Dr. Hector DeLuca . Rorcgfp mice were crossed with Rag1 KO mice to generate Rorcgfp Rag1 KO mice for cell sorting experiments of intestinal ILCs. VDRfl/fl mice were a gift from Dr. S. Kato  and were crossed with CD19Cre (B-VDR KO) or LckCre (T-VDR KO) mice. VDRfl/fl Cre+ (B-VDR KO or T-VDR KO) and their corresponding VDRfl/fl Cre- littermates (designated as WT) were used for experiments. Mice were fed purified diets made in the lab as described previously that differed only in vitamin D content - Rorc content . To gene content . For som content . The 1,2 content . Some miSerum 25(OH)D levels were measured by colorimetric assay using a 25(OH)D ELISA Kit , according to manufacturer's instructions. The limit of detection was 1.6 ng/mL 25(OH)D.C. rodentium strain ICC169  was a gift of Dr. Gad Frankel . C. rodentium was cultured overnight in Luria-Bertani broth with 50 μg/ml nalidixic acid . Mice were orally gavaged with 5 × 109 colony forming units (CFU) of C. rodentium in PBS. In some experiments, one dose of vancomycin  was given by oral gavage the day before C. rodentium infection. The vancomycin pretreatment increased infectivity without affecting the shedding kinetics of C. rodentium , IgG1and IgG2c (BD Biosciences).Detection of Colon LP cells were isolated as described previously and stained for flow cytometry . Brieflyhprt, β-actin, ifn-γ, il-17a, il-22, vdr, and rorc by real-time PCR. Total RNA was isolated from 1 cm distal colons of mice using RNeasy Mini kit . cDNA was synthesized by using AMV reverse transcriptase  and analyzed using the StepOnePlus real-time PCR system . Gene expression was normalized to Hprt or β-actin amplified and calculated with the ΔΔCt method. The primer sequences are listed in Supplementary Table A 1 cm sample of the distal colon was used to measure t test or two-way ANOVA with Bonferroni post-hoc tests were used for data analyses as indicated. The survival rates were compared by log-rank tests. For all the analyses, P values  were used to indicate significance. Values are the means ± SEM.Statistical analyses were performed with PRISM software by . Two-tailed Student's C. rodentium D . In the WT and T-VDR KO littermates there was no effect of genotype on the shedding of C. rodentium  or T cells (T-VDR KO) were infected with T Figure . There wT Figure . Howevern Figure . The C. m Figure . There wC. rodentium. Because there were no differences in C. rodentium susceptibility between D+ WT and D+ Cyp KO mice, the D+ Cyp KO group was eliminated. Before infection, the mRNA levels for ifn-γ, il-17, and il-22 were low and not different in D+ WT, D- WT, and D- Cyp KO mice  and D- Cyp KO (P < 0.0001) mice .ILC3 cells that produce IL-22 are critical in the survival and protection of Rag KO mice from odentium . D- Rag odentium . By d2 ps Figure . Only 1 s Figure . Converss Figure . The micC. rodentium infection. ILC3 cells express higher levels of mRNA for the VDR than other intestinal ILCs (Supplementary Figure in vivo could recover the ILC3 cell numbers, 1,25D treatments were started in D- mice at 6 weeks of age (Supplementary Figure C. rodentium shedding kinetics that were significantly different than both D+ WT and D-WT mice (significant group effect, Figure C. rodentium shedding kinetics that were more similar to D+ isotype treated than D- isotype treated mice (Figure C. rodentium in the feces (Figure C. rodentium (Figure C. rodentium clearance (Figure Experiments were done to determine whether ILC3, and/or IL-22 were important for the protective effects of vitamin D in host resistance to e Figure . Furthers Figure . TreatinC. rodentium infection was due to reduced frequencies of colonic ILC3 and ILC3-derived IL-22. IL-22 treatment of D- WT mice eliminated the need for vitamin D for the clearance of C. rodentium. In addition, treating D- Cyp KO mice with IL-22 prevented the lethality of the mice from C. rodentium. IL-22 secreting ILC3 are required in Rag KO mice for early survival from C. rodentium (C. rodentium infection as compared to only 14% of D+ Rag KO mice. IL-22 is a protective cytokine that regulates epithelial tight junction proteins to enhance gut epithelial integrity and induces antimicrobial factors in the small intestine to control the microbiota (The increased susceptibility of D- mice to odentium . The majcrobiota –33. D+ acrobiota . VitaminILC3 cells are vitamin D targets in the mouse colon. VDR mRNA levels were higher in mouse RORγt+ ILC3 cells from the colon than in other ILC cell populations (Supplementary Figure C. rodentium (Without antibiotic pretreatment, VDR KO mice were resistant to colonization with odentium . The colodentium  and Suppodentium  and Suppodentium . In the odentium . The vitin vitro and in vivo presumably by direct inhibition of IL-17 transcripts (il-17 mRNA, D- mice over expressed il17 mRNA in the distal colon following C. rodentium infection and the il17 mRNA was higher in the D- colon than the D+ colon. The higher Il17 mRNA in the D- colon (Figure C. rodentium, and therefore it seems unlikely that the failure of Th17 cells to expand following infection is a direct effect of vitamin D on Th17 cells. Instead, the failure of Th17 cells to expand in the D- mice following C. rodentium infection could be related to the reduced ILC3 frequencies. ILC3s express MHC class II molecules that present antigens to T cells (The Th17 cells in the colon of the D- mice failed to expand and there were significantly fewer Th17 cells in the d10 infected colon compared to D+ mice. 1,25D treatments have been shown to inhibit IL-17 nscripts , 39, 40.n Figure . T cell  T cells . ILC3 me T cells . ReducedCitrobacter-specific antibody response was lower in D- mice. B cells express the VDR and are vitamin D targets (Citrobacter-specific antibody responses and the B-VDR KO mice cleared the C. rodentium infection with the same kinetics as their WT littermates. Based on this data in B-VDR KO mice the effects of vitamin D on antibody responses to C. rodentium are likely indirect effects of vitamin D on B cells.The  targets . B-VDR K targets , 43. Con targets . Here B-C. rodentium infection. 1,25D treatment of D- mice that commenced at 3wks of age recovered the ILC3 frequencies in the colon. The 1,25D treatments and higher frequency of ILC3 cells improved the resistance of the D- mice to C. rodentium. Vitamin D was also needed to expand the infection induced Th17 response. IL-22 treatment of D- mice completely eliminated a need for vitamin D in the protection of D- mice from C. rodentium infection. ILC3 cells require vitamin D for either development or expansion and as a source of early IL-22. Vitamin D is a critical regulator of ILC3 cells and as a result D- mice have compromised resistance to C. rodentium infection of the gastrointestinal tract.Fewer ILC3, and less IL-22 resulted in the increased susceptibility of D- mice to Y-DL, SB, and MC conceptualized and designed the experimental studies. Y-DL, JA, KD, and SB performed the experiments and acquired and analyzed the data. Y-DL drafted the manuscript with the help of MC and SB critically revised the manuscript. All authors approved the publication of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


IL-1B polymorphisms rs16944 GG and rs1143627 AA and their diplotype GA/GA have been associated with significantly increased risk of intravenous immunoglobulin (IVIG) resistance in a Taiwanese population, but the relationship between rs16944 A/G and rs1143627 G/A and coronary artery lesions (CALs) in patients with KD has not been investigated. The present study is aimed at investigating whether the rs16944 A/G and rs1143627 G/A polymorphisms in IL-1B were associated with KD susceptibility and CALs in a southern Chinese population.  Kawasaki disease (KD) is a systemic form of self-limited vasculitis in children less than five years old, and the main complication is coronary artery injury. However, the etiology of KD remains unclear. The IL-1B, rs16944 A/G and rs1143627 G/A. According to the results, no significant association was observed between the IL-1B (rs16944 and rs1143627) polymorphisms and KD risk in the patients compared with the healthy controls in our southern Chinese population. However, in further stratified analysis, we found that children younger than 12 months with the rs16944 GG and rs1143627 AA genotypes of IL-1B had a higher risk of CALs than those with the AA/AG genotypes of rs16944 and GG/AG genotypes of rs1143627 .  We recruited 719 patients with KD and 1401 healthy children. Multiplex PCR was used to assess the genotypes of single nucleotide polymorphisms (SNPs), including two SNPs of IL-1B may significantly impact the risk of CAL formation in children younger than 12 months, which may contribute to the pathogenesis of KD. These findings need further validation in multicenter studies with larger sample sizes. Our results indicated that there was no association between the rs16944 A/G and rs1143627 G/A gene polymorphisms and KD susceptibility. However, the rs16944 GG and rs1143627 AA genotypes of  Kawasaki disease (KD) is characterized by systemic vasculitis and always occurs in children younger than 5 years. KD is also known as mucocutaneous lymph node syndrome . CoronarIL-1, TNF-α, IL-6, IL-8, and IL-10 [IL-1 family includes IL-1α, IL-1B, and IL-1Ra, which play fundamental roles in the inflammatory processes of KD. Two SNPs of IL-1B with functional implications have been reported, IL-1B rs16944 G and IL-1B rs1143627 A, and their effects on gene expression have been examined. IL-1B rs16944 G has been shown to have a relationship with increased transcriptional activity, rs1143627 A has been found to be related to reduce promoter activity, and the haplotype GA (rs16944 and rs1143627) has been associated with greater transcriptional activity than the other haplotypes. Weng et al. [IL-1B rs16944 GG and IL-1B rs1143627 AA genotypes or the GA/GA diplotype may be associated with initial IVIG treatment failure in Taiwanese children with KD, but no association with susceptibility to KD was observed. SNPs of IL-1B  or IL-1A-889 have been reported to have no significant association with KD susceptibility in Korean [Many studies have shown that immune activation and secretion of various cytokines play a key role in the pathogenesis of KD by mediating the imbalance of proinflammatory and anti-inflammatory responses. A variety of proinflammatory and anti-inflammatory cytokines have been reported to increase significantly during acute KD, such as nd IL-10 –14. Thesnd IL-10 –17. Studnd IL-10 . The IL-g et al.  demonstrn Korean , Iraniann Korean , or Taiwn Korean .IL-1B have been investigated in Korean, Iranian, and Taiwanese populations with KD, none has been examined in southern Chinese children with KD. The purpose of this study was to investigate the association of genetic polymorphisms in cytokine IL-1B rs16944 A/G and rs1143627 G/A with susceptibility to KD with or without CALs in southern Chinese children.Although polymorphisms in the proinflammatory cytokine A case control study was conducted on 719 patients with KD at Guangzhou Women and Children Medical Center in China, mainly between February 2013 and November 2017. The diagnosis of KD was based mainly on the Japanese diagnostic criteria . Simultaμl of whole blood per sample using a Genomic DNA Extraction Kit  according to the manufacturer's instructions. The concentration and quality of genomic DNA were measured using a nucleic acid quantifier, and the sample was stored at -80°C until later use. We performed multiplex PCR to genotype the SNPs, including rs16944 A/G and rs1143627 G/A. The primer sequences were as follows: rs16944: forward 5′-TAAATGGGTACAATGAAGGGCCA-3′, reverse 5′-CAATTTTCTCCTCAGAGGCTCCT-3′; rs1143627: forward 5′-TCGAAGAGGTTTGGTATCTGCC-3′, reverse 5′-GCTTCCACCAATACTCTTTTCCC-3′. Briefly, high-quality genomic DNA samples were genotyped by PCR using multiple gene-specific primer pairs to enrich the specific SNPs and indexing primers to enable massive parallel sequencing on the Ion Proton System (Life Technologies). For the specific procedures, please refer to our previous article [All collected experimental whole blood samples were thawed on ice, and DNA was extracted from 200  article . MoreoveIL-1B and KD susceptibility was evaluated by calculating the odds ratio (OR) and the 95% confidence interval (CI), and an unconditional univariate logistic regression analysis was performed. Adjusted ORs were calculated by multivariate analysis with adjustment for age and gender. All statistical analyses were conducted using SAS software , and P < 0.05 indicated statistical significance.The chi-square test was performed to evaluate the distributions of demographic variables and genotype frequencies in KD patients and controls. Hardy-Weinberg equilibrium (HWE) was calculated for samples by using the chi-squared goodness-of-fit test. The association between the rs16944 A>G and rs1143627 G>A polymorphisms of P = 0.147) and gender (P = 0.546). The mean ages were 28.96 ± 25.34 months for patients (range 1-166) and 28.05 ± 28.05 months for controls (range 1-144). Of the KD patients, 32.13% and 67.87% were female and male, respectively, and the controls were 33.55% female and 66.45% male. According to the American diagnostic guidelines, CALs were defined as coronary vessels with an internal diameter ≥ 2.0-3.0 mm in a child younger than 5 years of age or >4.0 mm in those 5 years of age and older [The clinical characteristics are summarized in IL-1B, rs16944 A/G and rs1143627 G/A, and their associations with KD risk are displayed in P = 0.120); the GG genotype (GG vs. AA) had an adjusted OR of 1.17 . Using the rs1143627 GG genotype as the reference, the AG variant genotype (AG vs. GG) had an adjusted OR of 1.21 , and the AA genotype (AA vs. GG) had an adjusted OR of 1.19 . Under the additive, dominant, and recessive models, there were no significant associations between the rs16944 A/G and rs1143627 G/A polymorphisms and KD susceptibility after adjusting for age and gender .The genotype distributions of the selected SNPs of IL-1B gene polymorphisms and susceptibility to CALs in KD. Overall, 312 CALs and 407 NCALs were successfully genotyped, as listed in P = 0.941; GG vs. AA: adjusted OR = 1.27, P = 0.277; additive model: adjusted OR = 1.13, P = 0.247; rs16944 dominant model AG+GG vs. AA: adjusted OR = 1.10, P = 0.616; recessive model GG vs. AA+AG: adjusted OR = 1.25, P = 0.179; rs1143627 AG vs. GG: adjusted OR = 1.01, P = 0.945; AA vs. GG: adjusted OR = 1.26, P = 0.291; additive model: adjusted OR = 1.13, P = 0.266; rs1143627 dominant model AG+AA vs. GG: adjusted OR = 1.09, P = 0.633; recessive model AA vs. GG+AG: adjusted OR = 1.25, P = 0.194) after adjusting for age and gender.We then assessed whether there were associations between the IL-1B gene polymorphisms and CALs in children with KD in stratified analyses considering age and gender .We further explored the association between r Tables  and 5. WIL-1B and KD susceptibility in southern Chinese children, as observed previously in Iranian and Taiwanese populations. We failed to find any significant association between the IL-1B (rs16944 and rs1143627) gene polymorphisms and the risk of CALs compared with NCALs in KD. However, in the stratified analysis, if the age of onset was 12 months or younger, we observed that carriers of the IL-1B rs16944 GG genotypes and IL-1B rs1143627 AA genotypes had a higher risk of CALs in KD than those carrying the IL-1B rs16944 AA/AG genotypes and IL-1B rs1143627 GG/AG genotypes.In the present study, our results revealed no association between the two selected SNPs in γ, tumor necrosis factor α, IL-27, IL-10, IL-6, IL-4, IL-2, and IL-1B, are increased significantly in the acute phase of KD [IL-4  and a negative association of the CT genotype at -590 with the risk of KD in an Iranian population. Data from studies in the Taiwanese population support the significant associations of the CC genotype and CC/CC diplotype at IL-10  with the risk of KD and a relationship of the G allele frequencies of IL-10 (-1082) gene polymorphisms with CAA development in KD [IL-6 (-636 C/G) [IL-6 promoter at +162 bp, +168 bp, and -594 bp [IL-4  [KD has been extensively studied in terms of etiology, pathogenesis, treatment, prognosis, and intervention factors, but the pathogenesis of KD has not been clearly elaborated –28. Abnose of KD , 30. Chase of KD  found a nt in KD , 33. The636 C/G) , the IL- -594 bp , and IL-IL-1 family includes IL-1α, IL-1B, and IL-1Ra, which play fundamental roles in the inflammatory processes of KD. Lee et al. [IL-1B regulates the development of CALs and is blocked by an IL-1 receptor antagonist. Furthermore, IL-1 levels may be influenced by IL-1 polymorphisms. Thus, IL-1A, IL-1B, and IL1RN are considered attractive candidate genes for vasculitis. IL-1B has been reported to be related to the functionality of SNPs within the gene, and IL-1B rs16944 and rs1143627 are essentially in complete linkage disequilibrium [IL-1B (rs16944 and rs1143627) did not correlate with the risk of KD and IVIG resistance, but the IL-1B rs16944 GG and rs1143627 AA genotypes or the GA/GA diplotype significantly increased the risk of IVIG resistance in Taiwanese children with KD. Assari et al. [IL-1B (rs16944 and rs1143634) polymorphisms and KD patients in an Iranian population. Furthermore, the results of a study by Kim et al. [IL-1B rs1143634 G/A is associated with genetic susceptibility to KD and that there is no significant difference in the frequency of this genotype between KD with CALs (n = 32) and KD without CALs (n = 77). In the present case control study, we repeated this exploration of IL-1B rs16944 and rs1143627 genetic polymorphisms and KD susceptibility, but we also investigated the association of these two SNPs with or without CAL formation in southern Chinese children with KD. We found that two SNPs of the IL-1B gene were not associated with KD or the development of CAL susceptibility, but in children less than 12 months of age, compared with carriers of the AA/AG and GG/AG genotypes, carriers of the IL-1B rs16944 GG and rs1143627 AA genotypes had a significantly increased risk of development of CALs (P = 0.0027), which may be ascribed to the fact that young children may be more genetically susceptible to KD risk. Additionally, the incidence of KD tends to be higher in children younger than 5 years of age. Moreover, according to the data from epidemiological studies, KD is an age- and gender-related disease that generally occurs in children aged <5 years and is more severe in children aged <12 months [IL-1B rs16944 A/G and rs1143627 G/A polymorphisms; other potential SNPs of IL-1B and potential mechanisms of polymorphisms were not considered and remain to be studied. Third, due to a lack of information on the living environment, exposure factors, and dietary intake, we analyzed only the relationship between IL-1B gene polymorphisms and susceptibility to CALs in this study.The e et al.  indicateilibrium . In geneilibrium  reportedi et al.  showed nm et al.  suggeste2 months , 40. HowIL-1B (rs16944 and rs1143627) gene polymorphisms and KD susceptibility or the formation of CALs, these SNPs may contribute greatly to the risk of CALs in southern Chinese children younger than 12 months of age. However, studies investigating the IL-1B rs16944 A/G and rs1143627 G/A polymorphisms with multicenter and larger populations are needed to confirm our results.In conclusion, although there was no association between


Rotavirus D (RV-D) is gaining importance as a cause of gastroenteritis and runting and stunting syndrome (RSS) in poultry. To date, information is scarce on the molecular analysis of RV-D isolates worldwide. In this study, the VP7 gene, a major constituent of outer capsid structural protein, from a RV-D isolate (UKD48) obtained from Uttarakhand state was analyzed. Phylogenetically, the RV-D isolate was found to be closely related to a South Korean strain, and the nucleotide percent identity varied from 80.4–84.2% with other RV-D strains available globally. Furthermore, domain investigation within 21 aligned amino acid sequences of the VP7 gene affirmed that this gene has several domains: a conserved glycosylation site (N–I–T) having an important role in protein folding; a N-terminal signal peptide (“ITG”) for endoplasmic reticulum retention; and two hydrophobic sites for elucidating transmembrane portions, antigenic structures, and so forth. The findings suggest that the VP7 gene of the Indian RV-D isolate is genetically distinct from those of other avian RV-Ds. Although biological evidence is still needed to prove the functional characteristics of these domains in outer capsid structural proteins, the present study adds new knowledge and derives the need for further investigation. Reoviridae, the RVs that infect birds, that is, avian rotaviruses (AvRVs), include A, D, F, and G species, which are classified on the basis of the antigenic properties of the VP6 protein. Among the four AvRVs, RV-A and RV-D exemplify high shedding frequencies. Apart from causing diarrhea, RV-D plays a key role in causing runting and stunting syndrome [VP7 gene of an RV-D isolate (UKD48) to other RV-D strains, N-glycosylation sites, distribution of hydrophobic/hydrophilic domains, and defines N-terminal signal peptide domains within the target gene sequences.Rotaviruses (RVs) have been identified as one of the main etiological agents of diarrhea and enteritis in mammals, including humans and avian species ,2. Belonsyndrome . The RV syndrome . VP7 is syndrome . HithertVP6 gene-based RT-PCR [VP7 gene analysis with amplification of the complete coding sequence of the VP7 gene using RT-PCR. Primers were designed  to amplify a fragment of the VP7 gene by identifying the consensus regions based on the nucleotide sequence alignment of VP7 gene sequences obtained from the NCBI database. The optimized PCR conditions include an initial cycle of 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 48 °C for 45 s, 72 °C for 1 min, and a final extension cycle of 68 °C for 1 min using DreamTaq PCR master mix .A sample  was collected from a young layer chick (UKD48) during postmortem from Uttarakhand state (India), and was subjected for the RV-D confirmation by using VP7 gene of RV-D available in the NCBI database were retrieved. The CLUSTALW program of the version 6 of MEGA software package was used for the pairwise and multiple sequence alignment of retrieved sequences along with the present study sequence (UKD48) [PCR-amplified product was resolved on 1.5% agarose gel, cloned into the pGEM-T vector system , and transformed. Plasmid was isolated using GeneJET Plasmid Miniprep Kit , and positive recombinant clones were sequenced by SciGenom labs . Twenty sequences for the VP7 gene [http://www.cbs.dtu.dk/services/TMHMM/) [http://www.sbg.bio.ic.ac.uk/phyre2/) [A dendrogram was constructed using MEGA6 (v6.05) using the maximum likelihood method with the Tamura 3+G+I model for the /TMHMM/) . To infephyre2/) .VP6 gene-based RT-PCR, yielding an expected amplicon of 185 bp  was amplified  exhibiting signs of severe enteritis was confirmed for the presence of RV-D using f 185 bp A. Subseqg RT-PCR B. In themplified B.VP7 gene sequences are available in the public domain, the majority of which are from Brazil. Thus, there is a need to exemplify the work on the genetic characterization of RV-D isolates from different parts of the world for understanding the virus’ evolution mechanisms.Evolutionary analysis of the UKD48 strain (accession no. KF142489) of RV-D revealed that the sequences fell into two distinct major clusters, wherein the present study sequence was found neighboring the South Korean RV-D strain D62 (KM254196) . NotablyApart from phylogenetic findings, functionally important domains in the aligned amino acid sequences were analyzed. It is interesting to note here that one N-glycosylation site, N–X–S/T (where X can be any amino acid except proline), was found conserved in the form of N–I–T (243–245) in all the RV-D sequences, which is in contrast with the earlier studies done by Trojnar and coworkers, where no N-glycosylation site was evident . GlycosyVP7 gene contains several important domains as seen in the case of mammalian RV-A, including one N-glycosylation site which plays a role in protein folding and cellular attachment. However, further biological studies are necessary to support this data.The findings of the current study, based on the low sequence identity and distinct clade in the phylotree, suggest that the Indian RV-D isolate (UKD48) is genetically divergent from other avian RV-Ds. Furthermore, the


Cell viability, proliferation, apoptosis, and migration were detected by Cell Counting Kit-8, BrdU assay, flow cytometry, and transwell two-chamber assay, respectively. The cell cycle-related protein cyclin D1 and cell apoptotic proteins  were analyzed by western blot. Alteration of H19 expression was achieved by transfecting with pEX-H19. PI3K/AKT pathway was measured by detecting phosphorylation of PI3K and AKT. SchA significantly decreased cell viability in a dose-dependent manner. Furthermore, SchA inhibited cell proliferation and cyclin D1 expression. SchA increased cell apoptosis along with the up-regulation of pro-apoptotic proteins  and the down-regulation of anti-apoptotic protein (Bcl-2). Besides, SchA decreased migration and down-regulated matrix metalloproteinases (MMP)-2 and MMP-9. SchA down-regulated lncRNA H19. Overexpression of H19 blockaded the inhibitory effects of SchA on A375 cells. SchA decreased the phosphorylation of PI3K and AKT while H19 overexpression promoted the phosphorylation of PI3K and AKT. SchA inhibited A375 cell growth, migration, and the PI3K/AKT pathway through down-regulating H19.Malignant melanoma (MM) is one of the malignant tumors with highly metastatic and aggressive biological actions. Schizandrin A (SchA) is a bioactive lignin compound with strong anti-oxidant and anti-aging properties, which is stable at room temperature and is often stored in a cool dry place. Hence, we investigated the effects of SchA on MM cell line A375 and its underlying mechanism. A375 cells were used to construct an  Malignant melanoma (MM) is one of the malignant tumors with highly metastatic and aggressive biological actions. Besides, MM accounted for 60–80% of skin-cancer related deaths . Recent Fructus schisandra, has been proven to play effective functions in various diseases due to its biological properties (Schizandrin A (SchA), extracted from operties . For exaoperties . SchA inoperties . Importaoperties . Hence, In recent years, long non-coding RNAs (lncRNAs) have received considerable attention due to their important functions in regulating gene expression in multiple approaches, such as chromosome remodeling, transcription, and post-transcriptional processing . Increasin vitro and investigated the effects of SchA on A375 cells and its underlying mechanisms.In our study, we used A375 cells to construct a MM model ® CRL-1619™) was purchased from American Type Culture Collection . The culture medium for A375 cells was Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum . The cells were maintained in the environment with 5% CO2 and 37°C.The MM cell line A375 (ATCCSchA (≥98.0% (HPLC), 5 cells/well, under proper conditions (37°C and 5% CO2). Then, 10 μL CCK-8 solution was added and cells were incubated for 1 h. After incubation, absorption was read at 450 nm using a Microplate Reader .Cell Counting Kit-8  was used for examining cell viability. Treated A375 cells were seeded in a 96-well plate at the density of 2×10Bromodeoxyuridine  was used for cell proliferation assay. In brief, A375 cells treated with SchA or co-treated with SchA and transfected with pEX-H19 were plated in a 96-well plate. Then, BrdU (1 mg/mL) was added to the cultured cells. Cells were then incubated for 3 h and proliferated cells were labeled. Finally, cells incorporated with BrdU were quantified using a BrdU cell proliferation assay kit .Propidium iodide (PI) and fluorescein isothiocyanate (FITC)-conjugated annexin V staining  were used for cell apoptosis assay. In brief, cells at the density of 100,000 cells/well were seeded in a 6-well plate. Treated cells were washed twice with precooled phosphate buffer saline (PBS) and resuspended in binding buffer. Then, 5 μL annexin V-FITC was added and mixed gently, and the mix put in the dark for incubation for 15 min. In addition, 5 μL PI was added to the sample. The apoptotic cell rate was measured with a flow cytometer .5 cells/mL) without serum was added to the upper transwell. Then, 600 μL culture medium with 10% FBS was added to the lower compartment of the 24-well transwell. A375 cells were maintained for 24 h at 37°C with humidified air containing 5% CO2. After incubation, cells at the upper surface of the filter were removed by a cotton swab, and the filter was fixed with methanol for 5 min. A375 cells at the lower surface of the filter were stained by Giemsa for 15 min. Cells were counted on a 100× microscope .Cell migration was evaluated by a modified two-chamber migration assay with a pore size of 8 μm. A cell suspension of 100 μL .To clarify the function of H19, pEX-H19 and its corresponding negative control (NC) pcDNA3.1  were transfected into A375 cells. Pre-treated cells at the density of 2×10® PrimeScript®PLUS RT-RNA PCR kit  was used for real-time PCR analysis to determine the expression level of H19. GAPDH was the internal control for H19.Total RNA was obtained from A375 cells using Trizol reagent (Invitrogen). The One-Step SYBRWestern blot was used in our study to detect protein expression. Protein was obtained from A375 cells using RIPA lysis buffer  supplemented with protease inhibitors (Thermo Fisher Scientific). The BCA™ protein assay kit  was used for determining proteins concentration. The western blot system was established by a Bio-Rad Bis-Tris Gel system following the manufacturer's instructions. Primary antibodies included: anti-cyclin D1 antibody (ab134175), anti-Bcl-2 antibody (ab32124), anti-Bax antibody (ab32503), anti-pro caspase 3 antibody (ab32150), anti-cleaved caspase-3 antibody (ab32042), anti-pro caspase-9 antibody (ab32539), anti-cleaved-caspase-9 antibody (ab2324), anti-matrix metalloproteinase (MMP)-9 antibody (ab73734), anti-MMP-2 antibody (ab37150), anti-phosphatidylinositol 3′-kinase (PI3K) antibody (ab191606), anti-phospho-PI3K antibody (ab182651), anti-protein kinase B (AKT) antibody (ab8805), anti-phospho-AKT antibody (ab8933), and anti-β-actin antibody (ab8227), all from Abcam (UK). Primary antibodies were prepared in 5% blocking buffer and diluted according to product instruction. These primary antibodies were incubated in membrane and maintained at 4°C overnight at the recommended concentration. For the second antibody incubation, cells were incubated with horseradish peroxidase (HRP) conjugated second antibody. Detection of signals and band intensity analyses were done by Image Lab™ Software .t-test. P values <0.05 were considered significant.Data are reported as means± SD, based on at least three experiments. Statistical analyses were performed using Graphpad 6.0 statistical software . P-values were calculated using a one-way analysis of variance (ANOVA) and Student's 50 under this concentration. BrdU assay was performed to detect cell proliferation and we found that SchA (30 μM) significantly decreased cell proliferation (P<0.01, Firstly, an experiment was designed to determine the concentration at which SchA had the best effect in decreasing cell viability. As shown in  P<0.01, . In addi P<0.01,  and enha P<0.01, . Moreove P<0.01, . Taken tIn MM, migration is one of the main approaches for tumor metastasis . Hence, Previous research has reported that lncRNA H19 has shown tumor-promoting effects in multiple tumors . In our To identify the mechanism by which SchA regulated cell proliferation and apoptosis, pEX-H19 was transfected into A375 cells. The significant up-regulation of H19 after transfection with pEX-H19 indicated high transfection efficiency P<0.01, . Then, wSimilarly, we detected the effect of H19 overexpression on cell migration. We found that co-treatment with SchA and H19 overexpression increased cell migration P<0.05,  as well PI3K/AKT was reported to be involved in MM . Hence, in vitro cell model and investigated the effects of SchA on A375 cells. Results demonstrated that SchA decreased cell proliferation and migration, increased cell apoptosis, and inhibited PI3K/AKT signaling pathway through down-regulating lncRNA H19.MM is a catastrophic skin cancer with aggressive biological actions, resistance to chemotherapy, and poor results . MM has in vitro model of MM (Cell line A375 is often used for the el of MM . Firstlyel of MM . Furtherel of MM ,20. Cyclel of MM . In our el of MM  that SchIn addition, it is well-known that apoptosis plays important roles in cancer cells, and apoptosis related proteins are considered a promising target for molecule-based pharmacological intervention . SchA inFurthermore, migration of cells to surrounding tissues is one of the initial and critical steps in the progression of cancer metastasis. Metastasis is the most important cause of cancer-related death . TherefoIncreasing evidence suggested that lncRNAs acted as molecular switches to regulate cell proliferation, cell apoptosis, and cell migration . Among tTo alter the expression of H19, pEX-H19 was transfected into A375 cells. The results showed that co-treatment and transfection with pEX-H19 and SchA blockaded the effects of SchA on A375 cells as evidenced by increasing cell proliferation and migration, and inhibiting cell apoptosis. This is consistent with previous findings that H19 enhances breast cancer cell proliferation  and elevPI3K/AKT signaling pathway has been reported to play important roles in cell proliferation, survival, and metabolism , and wasOur study demonstrated that SchA inhibited proliferation and migration of melanoma A375 cells, and increased apoptosis and inhibited PI3K/AKT pathway through down-regulation of lncRNA H19. Our study may lay the foundation for the study of MM in the future.


SGK1) and the tumor metastasis–suppressor gene N-Myc downstream regulated gene 1 (NDRG1) are up-regulated and that the microRNAs miR-29a and miR-101-1 targeting the 3′-UTR of SGK1 are down-regulated in response to progesterone. We further demonstrate a dual-phase transcriptional and post-transcriptional regulation of SGK1 in response to progesterone, leading to an up-regulation of NDRG1 that is mediated by a set of genes regulated by the transcription factor AP-1. We found that NDRG1, in turn, inactivates a set of kinases, impeding the invasion and migration of breast cancer cells. In summary, we propose a model for the mode of action of progesterone in breast cancer. This model helps decipher the molecular basis of observations in a randomized clinical trial of the effect of progesterone on breast cancer and has therefore the potential to improve the prognosis of breast cancer patients receiving preoperative progesterone treatment.Preoperative progesterone intervention has been shown to confer a survival benefit to breast cancer patients independently of their progesterone receptor (PR) status. This observation raises the question how progesterone affects the outcome of PR-negative cancer. Here, using microarray and RNA-Seq–based gene expression profiling and ChIP-Seq analyses of breast cancer cells, we observed that the serum- and glucocorticoid-regulated kinase gene ( PR in breast cancer cells  and N-Myc downstream regulated gene 1 (NDRG1), along with down-regulation of miR-29a and miR-101-1, predicted to bind the 3′-UTR of SGK1, independent of the hormonal receptor status of the cells . The up-regulation of SGK1, known to harbor multiple progesterone response elements (PREs) . Also, knockdown of PR significantly decreased the progesterone-induced up-regulation in expression of SGK1 in PR-positive cells . Moreover, SGK1 showed an increased expression based on analysis of the RNA-Seq data, reported earlier , along with differentially enriched binding of PR and p300 at the SGK1 loci in response to progesterone treatment, based on ChIP-Seq data , in T47D and MCF7 PR-positive cells . Interestingly, SGK1 activation (induction of p-SGK1 in response to progesterone) was found to be comparable in breast cancer cells, regardless of their PR status (C). Next, we validated SGK1 as a target of miR-29a and miR-101-1 by co-expressing the microRNAs along with firefly luciferase reporter genes cloned upstream to 3′-UTR of SGK1. Ectopic expression of both of the microRNAs decreased the firefly luciferase activity in 293FT cells expressing the 3′-UTR of SGK1. Consistent with the findings, transfection with anti-miRs targeting miR-29a and miR-101-1 not only rescued the repression of luciferase activity in 293FT cells (A) but also led to sustained expression levels of SGK1 based on Western blot analysis of breast cancer cells (B). Taken together, the data suggest convergence of the dual mode of regulation at SGK1 in response to progesterone treatment, along with up-regulation of NDRG1 in multiple breast cancer cell lines independent of their PR status.An integrated analysis of microarray-based mRNA expression profile and deep sequencing of noncoding small RNA of breast cancer cells  led us to identify up-regulation of a serum- and glucocorticoid-regulated kinase gene (s (PREs) , 21, andesterone , A–D. Al earlier  , which in turn led to a significant reduction in cell migration and cell invasion (B and C). In a reciprocal approach, depletion of SGK1 in T47D and MDA-MB-231 cells led to a decrease in expression of NDRG1 (A) with an inverse effect observed on migration and invasion of the breast cancer cells (B and C), regardless of progesterone treatment (A and B). Furthermore, consistent with the genetic perturbation, pharmacological inhibition of SGK1 with 1 μm GSK650394A similarly blocked the effect of progesterone on breast cancer cell migration and cell invasion, suggesting an essential role of the SGK1/NDRG1 axis downstream to progesterone in breast cancer cells independent of their hormonal receptor status  and EGR1 in response to stress-induced activation of kinases such as p38, JNK, and ERK ). Consistent with this finding, knockdown of SGK1 significantly reduced the expression of AP-1 network genes (C and D), and depleting the expression of an AP-1 network gene, EGR1, abrogated the expression of NDRG1 (A), a downstream component of the pathway, in T47D and MDA-MB-231. Taken together, these results suggest that progesterone and SGK1 regulate the expression of NDRG1 via the AP-1 network genes. and ERK –25. We rand ERK –. Thus, wrk genes , C and Dof NDRG1 A, a downNDRG1 mediates inactivation of EGFR/AKT1/ERK1/2 kinases in response to progesterone, we knocked down the expression of NDRG1 in T47D and MDA-MB-231 cells (A and 11A). Interestingly, two of three shRNA clones targeting NDRG1 significantly increased the phosphorylation of EGFR (Tyr-1068), AKT (Ser-473), and ERK1/2 (Thr-202/Tyr-204) (B and 11B), which remained unaffected even upon treatment with progesterone, suggesting an essential role of NDRG1 to mediate progesterone response (A and B) and S4 (A and B)Figs. S3 . Furthermore, breast cancer cells expressing constructs targeting EGR1 and NDRG1 displayed an increase in breast cancer cell migration . Also, the NDRG1-depleted cells continued to show an increased cell migration regardless of progesterone treatment . Taken together, our results suggest that the SGK1/NDRG1 axis mediates regulation of activation of kinases involved in breast cancer cell migration, independent of their hormonal receptor status.We recently showed that progesterone decreases the activation of multiple kinases like EGFR, AKT1, and ERK1/2 in breast cancer cells, leading to suppression of cell migration . To test31 cells A and 11ATyr-204) B and 11BEGFR Tyr-68, AKT (SGK1), predominantly driven as a direct transcriptional target, consistent with earlier reports  mediates the effect of progesterone by regulating the expression of SGK1 and NDRG1 and cell migration (PGRMC1 and SERBP1), which are uniformly expressed across breast cancer cells . Cells were tested for mycoplasma and were made mycoplasma-free using EZKill mycoplasma removal reagent (HiMedia). T47D, BT474, MDA-MB-231, MCF7, and ZR-75-1 cells were cultured as described previously . The cell lines were authenticated by DNA short tandem repeat profiling using the Promega GenePrint 10 system, and analysis was done using the GeneMarker HID software and the ATCC database  was added to the cells for 2 h, followed by 10 nm progesterone treatment for 6 h in the same medium. An equal amount of alcohol was used as vehicle control. The treatment conditions for progesterone and mifepristone were standardized based on the expression change of three known candidate genes .Breast cancer cells were treated with progesterone, and RNA isolation was performed as described earlier , 36. AddFig. S5). All possible comparisons were taken into consideration as progesterone versus control (P versus C), mifepristone + progesterone versus control (M+P versus C), and progesterone versus mifepristone + progesterone (P versus M+P). From our analysis, we identified 623, 553, 1873, 532, 1764, and 4703 differentially expressed genes in T47D, MCF7, BT474, ZR-75-1, MDA-MB-231, and 184A1 breast cell lines, respectively. The microarray raw data are available on ArrayExpress (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-6742/)Gene expression profiling was performed using the BeadChip Illumina microarray platform. Raw data (.idat files) of the BeadChip Illumina platform were converted to readable format using Genome studio software (version V2011.1). Probe level data were converted into gene-centric estimates and used for further processing. The Bioconductor lumi package was used for preprocessing the data, which includes quality control steps, background correction, normalization, log transformation, etc. and finally differential gene analysis. Robust spline normalization and variance stabilization transformation methods were used for normalization and transformation, respectively. To specifically select for highly variable genes, unexpressed, nonannotated, and false positive genes were excluded from the analysis. Median absolute deviation was carried out to make samples comparable with each other. The differential gene expression cut-off was set as 0.5 ≤ log (-fold change) ≤ −0.5 in at least one of the three conditions . Heat maps were constructed using MeV software (version 4.9.0) (ChIP-Seq data for PR and p300 pulldown in PR-positive breast cancer cell lines (MCF7 and T47D) was obtained from the gene expression omnibus  GSE6835 using BWAnalysis of RNA-Seq–based gene raw counts for T47D and MCF7 cell lines, untreated and treated with progesterone (obtained from GSE68358), was performed using R language. DESeq2 bioconductor package  was usedSGK1 and NDRG1 were found to be the top up-regulated and recurrent gene in response to progesterone across multiple cells . Next, we analyzed our small RNA-Seq data to identify differentially expressed microRNA in response to progesterone treatment predicted to bind to the 3′-UTR of the SGK1 gene using six different microRNA binding site prediction tools  was used. Untransfected parent cells were used as control for overexpression. Positive clones were selected using 1 μg of puromycin. Overexpression experiments were performed in T47D (PR-positive) and MDA-MB-231 (PR-negative) breast cancer cells. For the knockdown of SGK1, NDRG1, and EGR1 genes in T47D and MDA-MB-231 cells, three lentiviral shRNA constructs  each, against these genes, were used for genetic depletion. Positive clones were selected using 1 μg of puromycin. For transient knockdown of the progesterone receptor (PR or NR3C3), three lentiviral shRNAs targeting the PR were used. 48 h post-transduction, T47D cells, without selection, were used for further experimentation. The short hairpin-nontargeting (sh-NT) was used as vector/scrambled control for all knockdown experiments.For overexpression of GR (NR3C1) was performed in PR-negative MDA-MB-231 cells. Two siRNAs targeting GR were used, along with one siRNA-control  to compare the expression of GR and downstream targets. Following siRNA transfection for 72 h using Lipofectamine RNAiMAX (Thermo Fisher Scientific), cells were treated with progesterone as detailed above and processed for RNA isolation and protein sample preparation.A transient siRNA-mediated knockdown of the Protein samples were prepared, and Western blots were developed as described earlier . Brieflym concentration of GSK650394A  was added to the respective wells for 4 h, as discussed in other studies (m progesterone for 6 h and then used for RNA or protein isolation.Cells were grown until 70–80% confluence in a 6-well dish and then serum-starved using low-glucose DMEM (HiMedia) for 24 h. 1.0 μ studies . After 4Cell invasion assay was performed as described earlier . BrieflyWound scratch migration assay and analysis was performed as described earlier . BrieflySGK1 3′-UTR was performed as described earlier (miR-29a and miR-101-1 containing the seed sequence was PCR-amplified using genomic DNA isolated from T47D. Amplicons were cloned in a T/A cloning vector (Fermentas), followed by subcloning in BamHI and HindIII sites of pCDNA 3.1(−) expression vector (Invitrogen). SGK1-3′-UTR of 1000 bp was PCR-amplified using T47D cDNA. Amplicons were cloned in a T/A cloning vector followed by subcloning between XbaI sites in a pGL3-promoter vector . For the Dual-Luciferase assay, 293FT cells  were transfected using Lipofectamine 2000 reagent (Thermo Fisher Scientific) with a combination of these constructs along with a Renilla luciferase vector  in separate wells. 5 nm miR inhibitors (anti-miRs) (Sigma) were also transfected in combination to expression vectors for specifically inhibiting the activity of both of the microRNAs. 48 h post-transfection, cells were lysed, and a luciferase assay was performed to measure firefly luciferase activity after normalization to Renilla luciferase values . The experiment was performed in triplicates, and differences between groups showing p values of <0.05  were considered significant.Cloning of microRNA sequences and  earlier . Brieflym negative control miR inhibitor (anti-miR-129-2), anti-miR-29a, and anti-miR-101-1 (Sigma). Post-transfection, cells were incubated for 48 h and then treated with progesterone for 6 h as described above. Cell lysate was prepared, and Western blot analysis was performed to study expression of SGK1.T47D cells were grown up to 60% confluence and transfected with 25 nt test was used to determine the statistical significance.Statistical analysis was performed using GraphPad Prism version 5 software . Student's unpaired M. G. and A. D. designed the experiments. M. G., T. T., K. P., B. D., N. Y., S. J., N. G., K. T., P. T., S. D., R. P., H. D., S. S., K. K., D. K., and P. C. performed the experiments. S. D., S. G. and R. A. B. provided the reagents. M. G. and A. D. wrote the paper.


Ostreococcus tauri has three SSIII isoforms. In the present study, we describe the structure and function of OsttaSSIII-B, which has a similar modular organization to SSIII in higher plants, comprising three putative starch-binding domains (SBDs) at the N-terminal region and a C-terminal catalytic domain (CD). Purified recombinant OsttaSSIII-B displayed a high affinity toward branched polysaccharides such as glycogen and amylopectin, and to ADP-glucose. Lower catalytic activity was detected for the CD lacking the associated SBDs, suggesting that they are necessary for enzyme function. Moreover, analysis of enzyme kinetic and polysaccharide-binding parameters of site-directed mutants with modified conserved aromatic amino acid residues W122, Y124, F138, Y147, W279, and W304, belonging to the SBDs, revealed their importance for polysaccharide binding and SS activity. Our results suggest that OT_ostta13g01200 encodes a functional SSIII comprising three SBD domains that are critical for enzyme function.Starch is the major energy storage carbohydrate in photosynthetic eukaryotes. Several enzymes are involved in building highly organized semi-crystalline starch granules, including starch-synthase III (SSIII), which is widely conserved in photosynthetic organisms. This enzyme catalyzes the extension of the α-1,4 glucan chain and plays a regulatory role in the synthesis of starch. Interestingly, unlike most plants, the unicellular green alga  Starch plays a key role in the life cycle of plants as the primary form of carbohydrate storage for chemical energy. Additionally, it has acquired immense value as a vital source of energy and as a precursor of new biodegradable materials . Starch There are four biochemical steps in starch synthesis  carried out by different enzymes . In greeOstreococcus tauri dates from 1994 in a lagoon in Thau, France. This picoalga is a member of the Prasinophyceae, which places it in a key phylogenetic position as a study model because it shares the common ancestor of Chorophyta that currently dominate terrestrial photosynthesis. O. tauri lacks a cell wall and flagella, and contains a large nucleus, a single chloroplast, a mitochondrion, and a Golgi apparatus in a reduced cytoplasmic compartment  than any other algae or plant species   of which only SSIII-C has been characterized  . Thus, tcterized . Interesechanism .A. thaliana (ArathSSIII), containing a GT5 domain1 at its C-terminal end and three in tandem SBDs belonging to the CBM53 family in its N-terminal region (designated D1-D2-D3), demonstrated the importance and functionality of an SBD in a biosynthetic enzyme  domain, while the N-terminal region includes starch-binding domains (SBD), a specific group of carbohydrate-binding modules (CBM). The generic term CBM refers to a contiguous amino acid sequence with a distinctive fold within an enzyme that may or may not be catalytically active with carbohydrate substrates . The abic enzyme , 2011. TEscherichia coli. Our results show that the OsttaSSIII-B isoform is a modular protein with three N-terminal SBDs and a C-terminal CD, being the SBDs important for its catalytic activity. The purified recombinant protein displayed a high affinity toward branched polysaccharides such as glycogen and AP. Analysis of enzyme kinetic and polysaccharide-binding parameters of OsttaSSIII-B site-directed mutants with altered conserved aromatic amino acid residues W122, Y124, F138, Y147, W279, and W304, belonging to the SBDs, revealed their importance for polysaccharide binding and SS activity.Herein, we describe the cloning and expression of the OsttaSSIII-B gene, and purification and characterization of the recombinant protein produced in Escherichia coli strains XL1 Blue ]) and BL21 (DE3) pLysS  were purchased from Stratagene . Bacterial cells were grown at 37°C in Luria Bertani (LB) medium containing the appropriate antibiotics.Domains of OsttaSSIII-B were predicted using the KEGG Sequence Similarity Database (SSDB) Motif Search server . AlignmePaenibacillus polymyxa (PDB code: 2LAA), CBM25 from Bacillus halodurans amylase (PDB code: 2C3V), and the glycogen-binding domain of the AMP-activated protein from Rattus norvegicus (PDB code: 1Z0N), respectively  was cloned from O. tauri genomic DNA (kindly provide by Dr. Evelyne Derelle) into KpnII and HindIII restriction enzyme sites of the pRSET-C vector  using standard molecular biology procedures and primers OtSSIII-B Nt Fwd (TGCGGTACCCGCGTCGGTGGAG) and OtSSIII-B Ct Rev (CTAAGCTTTTACCGACCGATCATC). OsttaSSIII-B CD was amplified from pRSET-C::OsttaSSIII-B using primers OtSSIII-B CD Fwd (AAGGTACCTCTTCACGTCTGTCAC) and OtSSIII-B Ct Rev, and cloned into KpnII and HindIII sites of pRSET-C (KpnI and HindIII sites are underlined). The expression vectors pRSET-C::OsttaSSIII-B and pRSET-C::OsttaSSIII-B CD  were used to transform E. coli BL21 (DE3) pLysS, and cells were grown at 37°C for 3 h, then induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated at 20°C for at least 18 h. Cells were harvested by centrifugation at 5000 × g for 15 min at 4°C, and the pellet was resuspended in 20 mM TRIS-HCl (pH 7.5). Cells were disrupted by sonication and centrifuged at 12,000 × g for 15 min at 4°C. The homogenate was loaded onto a HiTrap chelating HP column  equilibrated with binding buffer . The column was washed with 10–15 volumes of binding buffer, and each protein was eluted using a linear gradient of binding buffer and elution buffer  (The OsttaSSIII-B gene (idazole) . The preGCGGTGGCCGCCGATGTGAACGTG; OtB F138A, CGCGCGTTCTCGATGGATGCCGTGTTCAAGGGAGA; OtB Y147A, GAGACGGCGAGGCGGCCGAGAAGGACGAAG; OtB W279A, AGAACAACGACTGGGCGGTCGCCGACGTCC, and OtB W304A, CGTCGA AGAGTCGGCCGCGGATAACAACGAACAG. Mutations were confirmed by DNA sequencing . The resultant plasmids, named pRSET-C::OsttaSSIII-B W122AY124A, pRSET-C::OsttaSSIII-B F138A, pRSET-C::OsttaSSIII-B Y147A, pRSET-C::OsttaSSIII-B W279A, and pRSET-C::OsttaSSIII-B W304A  were used to transform E. coli BL21 (DE3) pLysS cells. Mutated recombinant proteins were expressed, purified, and stored at −40°C as described above.OsttaSSIII-B mutants W122AY124A, F138A, Y147A, W279A, and W304A were obtained using the QuickChange II site-directed mutagenesis kit . The pRSET-C::OsttaSSIII-B vector was used as template for PCR amplification. The following primers (and their complements) were used : OtB W122AY124A, GGTGAAGGGGACGTCGDifferential scanning fluorimetry (DSF) was performed on an Applied Biosystems real-time PCR instrument . The tecE. coli alkaline phosphatase , 0–1 mM ADPGlc and one of the following polysaccharides: 0–2 mg/ml rabbit liver glycogen (G8876 Sigma-Aldrich), 0–3 mg/ml potato AP , 0–1 mg/ml potato AM , or 0–2 mg/ml potato starch . Reaction vessels were gently shaken all over the assay to keep substrates in suspension. When UDPGlc was used as the glycosyl donor, all other assay conditions were identical, but ADPGlc was replaced with UDPGlc in the range of 0–10 mM. After incubation for 20 min at 30°C, the reaction was stopped by the addition of Malachite Green–ammonium molybdate (MG–am) reagent. This MG–am solution was prepared by merging three volumes of 0.5 mM aqueous solution of Malachite Green  and one volume of 0.034 M ammonium molybdate in 4 M HCl. The color reaction was developed by mixing samples (1.0 volume) with the color reagent (6.7 volumes). The complex formed from the released Pi was measured at 630 nm as described (The activity of WT and mutated OsttaSSIII-B proteins was determined following the formation of inorganic phosphate (Pi) after hydrolysis of generated ADP by alkaline phosphatase by colorimetric assays as previously reported . The reaescribed .Vmax, S0.5 and Hill number (nH)] were determined from a non-linear fit of the Michaelis–Menten equation to the observed enzyme activities using GraphPad Prism v.6.01. The average values of at least three determinations ± SE are reported. Total protein concentration was determined using the Bradford method (Enzyme kinetic data were plotted as specific activity (μmol/min/mg protein) versus substrate concentration. All kinetic parameters [d method .A pH profile of OsttaSSIII-B activity was obtained by measuring activity at 30°C and pH values ranging from 6.0 to 10.0 in 50 mM BICINE buffer. The temperature profile of OsttaSSIII-B activity was followed by measuring activity at pH 8.0 and temperature values ranging from 20 to 45°C in 50 mM BICINE buffer. Both experiments were run with 2 mg/ml glycogen and 0.5 mM ADPGlc. Standard kinetic parameters were determined at pH 8.0 and 30°C.Kad) was measured as described previously with minor modifications  was determined from the slope as previously reported ; the glycogen-binding domain (GBD) of an AMP-activated protein from R. norvegicus; CBM25 from B. halodurans amylase; and CBM25 beta/alpha-amylase from P. polymyxa. These structures were used as templates for homology modeling. The starch binding sites described for ArathSSIII D2 are highly conserved in OsttaSSIII-B SBDs, specifically residues G335, W340, and Y394 (ArathSSIII numbering) belonging to binding site 1  amino acid sequence was also performed with the SSDB Motif Search server. We found that half of the CD has a structure similar to the GT5 family (Pfam 343 PF08323), while the other half has a structure similar to GT1 (Pfam PF00534), with relatively low It was not possible to construct a homology model of the full-length protein due to lack of a crystal structure of a protein with the same structural organization as SSIII, with three N-terminal SBDs and one C-terminal CD. Instead, we have built homology models of the individual SBDs  see Fig. DespiteDNA fragments encoding full-length OsttaSSIII-B and truncated OsttaSSIII-B CD were cloned into the pRSET-C vector for expression of proteins with N-terminal His-tags. To analyze the functions of the aromatic residues W122, Y124, F138, Y147, W279, and W304 in OsttaSSIII-B polysaccharide binding and enzymatic activity, the modified proteins OsttaSSIII-B W122AY124A, F138A, Y147A, W279A, and W304A were obtained by site-directed mutagenesis. Western blot assays revealed that the size of the mutated proteins was comparable to that observed for the WT enzyme Figure , with a S0.5 values for the acceptor polysaccharide were 0.08 mg/ml for glycogen, 0.12 mg/ml for starch, 0.08 mg/ml for AM, and 1.04 mg/ml for AP. By contrast, the S0.5 value for ADPGlc was 90 μM when glycogen was used as the acceptor substrate   . On the S0.5 for amylose than for starch or amylopectin . The lower specific activity observed with starch may be due to an inhibitory effect of the amylose present in this polysaccharide. It should be noted that OsttaSSIII-B showed a lower Similar results, demonstrating highest activity with glycogen as acceptor substrate, were previously reported for ArathSSIII  and OsttVmax value than that of the full-length enzyme, whereas the S0.5 value for ADPGlc was similar. The S0.5 value for the acceptor polysaccharide was twofold higher than the full-length protein , are highly conserved in ArathSSIII D2   (Kad = 3.38 ml/g) but the absence of SBDs could cause the lost of the binding capacity to starch and AP  are essential for binding to physiological substrates. Analysis of the spatial position of Y147 in our OsttaSSIII-B model shows that this residue is far away from the other two conserved binding amino acids in D1 (W96 and W105), suggesting that Y147, which aligns with binding site 1c in the D1 domain, is not involved in starch binding, and that D1 likely utilizes another aromatic residue in this position. Interestingly, some OsttaSSIII-B-mutated proteins displayed an increased polysaccharide binding capacity relative to the WT enzyme. The Kad values for AP of W279A and W304A variants were 2.6- and 1.5-fold higher than those observed for the WT protein   , and alsctively) . The higAP Table . Meanwhi in D1 W9 and W105Taken together, these results suggest that the AM specificity of the WT enzyme can be attributed to the binding residues in the D2 domain, while the ability to bind AP is related to the D1 domain. However, it is important to emphasize that amino acid residues in both D1 and D2 are essential for binding to starch. This is the first experimental evidence demonstrating the importance of amino acid residues in the D1 domain in the binding capacity of a SS enzyme.A. thaliana. Herein, we analyzed the contribution of several residues from D1 and D2 domains, representing the first characterization of the effects of both domains on the entire protein.A better understanding of the properties of CBMs and SBDs and their interactions with catalytic domains could provide new strategies to develop enzymes with improved catalytic efficiency toward polysaccharide substrates. In previous work, we evaluated the effects of mutating several residues in the D2 domain of SSIII from The adsorption parameters of the full-length protein for AM were twice higher than that obtained for the D123 domain alone , indicatA. thaliana SSIII. However, the W304A mutation  caused a 10-fold decrease in S0.5 for glycogen, indicating a greater contribution of binding site 1 in the apparent affinity for polysaccharide substrates.Finally, we evaluated the contribution of the two binding sites in the D2 domain, and observed that the W279A mutation moderately decreased the apparent affinity for glycogen, similarly to the W366 mutation in the D2 domain of JB planned and performed the experiments, analyzed the data, and wrote the manuscript. MBV performed the experiments and analyzed the data. NP performed the structure modeling and analyzed the data. DFG-C, AAI, MAB, and MVB planned the experiments, analyzed the data, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.