These enhancers potentiate the ubiquitylation of mutant β-Catenin by β-TrCP in vitro and induce the degradation of an engineered mutant β-Catenin in a cellular system. Distinct from PROTACs, these drug-like small molecules insert into a naturally occurring PPI interface, with contacts optimized for both the substrate and ligase within the same small molecule entity. The prospective discovery of ‘molecular glue’ presented here provides a paradigm for the development of small molecule degraders targeting hard-to-drug proteins.Protein–protein interactions (PPIs) governing the recognition of substrates by E3 ubiquitin ligases are critical to cellular function. There is significant therapeutic potential in the development of small molecules that modulate these interactions; however, rational design of small molecule enhancers of PPIs remains elusive. Herein, we report the prospective identification and rational design of potent small molecules that enhance the interaction between an oncogenic transcription factor, β-Catenin, and its cognate E3 ligase, SCF Directed protein degradation to target hard-to-drug proteins is a promising therapeutic approach. Here, the authors report the prospective discovery of small molecule “molecular glue” that inserts into a naturally occurring E3 ligase-substrate interface leading to degradation of substrate protein. These include immunosuppressants cyclosporine, FK506 and rapamycin as well as anti-cancer agents lenalidomide, pomalidomide, and thalidomide (collectively referred as immunomodulatory drugs or IMiDs). These IMiD molecules work by binding to the CRL4 E3 ligase substrate receptor protein, Cereblon (CRBN) and promoting interaction with the non-native lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IFZF3), as well as Casein kinase 1α (CK1α) leading to their ubiquitylation by RING E3 ligase CRL4CRBN and subsequent degradation by the 26S proteasome6. This degradation contributes to the clinical efficacy of IMiDs. Although thalidomide was discovered over 70 years ago, the molecular mechanism of IMiDs as PPI enhancers were discovered only recently following careful characterization of their pharmacologically relevant targets8. In addition to the IMiDs, other ligase-binding molecules are known in plant physiology. In particular, the small molecule hormone auxin, also known as indole-3-acetic acid (IAA), regulates plant growth and development by modulating the recognition of Aux/IAA transcriptional repressors by the SCFTIR1 E3 ligase9. Auxin binds to the substrate receptor protein TIR1 and promotes the interaction between TIR1 and the Aux/IAA substrate10. Importantly, auxin enhances the substrate interaction with TIR1 by extending the protein interaction interface for substrate binding, acting as a molecular glue by forming a continuous hydrophobic core for the TIR1 and substrate interaction10.Protein-protein interactions (PPIs) are central to most signaling pathways in cells and represent important nodes for therapeutic intervention. Although most small molecule PPI modulators are inhibitors, a small subset of PPI modulators are known to work by stabilizing PPIs14. These bifunctional molecules consist of two small molecules connected by a linker. One small molecule binds a protein of interest (substrate) and the other binds an E3 ubiquitin ligase, thereby bringing the substrate in close proximity with the ligase, promoting its ubiquitylation and degradation. Although PROTACs represent important new chemotypes to induce substrate degradation, the molecular weight, physiochemical, and pharmaceutical properties fall outside of the typical range for small molecule drugs potentially leading to challenges in drug development. These challenges might be circumvented by discovering and rationally designing smaller molecular glue-like molecules that bind both the substrate (either native-substrate or neo-substrate) and the ligase without the need for a linker to induce substrate degradation.The use of PPI enhancer molecules as ‘molecular glue’ to potentiate substrate:ligase interactions could be a therapeutically useful modality to employ against drug targets previously considered undruggable. However, rational design of such small drug-like molecules to degrade proteins that are prospectively identified remains elusive. In recent years, significant progress has been made to target protein degradation by employing hetero-bifunctional molecules 15. One such example is the Wnt signaling effector protein, β-catenin that is often dysregulated and stabilized in cancer17. In normal resting cells, the cytoplasmic pool of β-catenin is maintained at low levels through a process involving phosphorylation, ubiquitylation, and subsequent degradation. β-catenin is phosphorylated by the cytoplasmic destruction complex (DC) consisting of the tumor suppressor proteins, Axin and Adenomatous Polyposis Coli (APC), and two serine-threonine kinases, glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). β-catenin is initially phosphorylated at Ser45 by CK1, followed by GSK3 phosphorylation at Thr41, Ser37, and Ser3321. However, the sequential nature of the β-catenin phosphorylation cascade is not essential in certain colorectal cancer cells where phosphorylation at Ser45 is not required for phosphorylation at residues Ser33, Ser37, and Thr4122. The phosphorylated Ser33 and Ser37 in β-catenin are part of the phosphodegron sequence (DpSGφXpS) that binds to the F-box-containing E3 ubiquitin ligase protein β-TrCP, leading to β-catenin ubiquitylation by the SCFβ-TrCP cullin-RING ligase and subsequent degradation by the 26S proteasome23. In the majority of colorectal cancers, β-catenin is stabilized either by inactivating mutations to APC or Axin, which prevent proper β-catenin phosphorylation or by mutations within the β-catenin phosphodegron sequence itself25. While disruption of β-catenin phosphorylation frequently accounts for elevated protein levels, mutations in β-TrCP are rarely observed26. These β-catenin phosphodegron mutations including Ser33 and Ser37, impair the ability of β-catenin to effectively bind to β-TrCP leading to its stabilization and thereby facilitating an enhanced oncogenic transcriptional program. Despite being an important biological target, no small molecule inhibitors of β-catenin are available as therapeutic agents27. Therefore, using the concept of molecular glue, we sought to prospectively discover small molecule β-catenin degraders that work by restoring mutant β-catenin binding to β-TrCP. This molecular glue strategy represents a unique approach to target tumors with elevated levels of β-catenin. More broadly, it represents a therapeutic strategy to target proteins defective in ligase binding, ubiquitylation and degradation that have been implicated in various diseases28.Transcription factors remain extremely challenging proteins to target, despite being implicated in multiple diseasesIn this study, we report the prospective identification and rational structure-guided optimization of potent molecular glue-like small molecules intentionally designed to enhance the PPI between mutant β-catenin and β-TrCP that potentiate cellular degradation of an engineered mutant β-catenin in a cellular system. This work establishes a paradigm for rational design of small molecule enhancers to target ubiquitylation and degradation of proteins, including oncogenic transcription factors, by harnessing the ubiquitin proteasome system.24. These mutations at Ser37 impair the ability of monophosphorylated Ser33 β-catenin peptides to effectively bind β-TrCP by up to 300-fold compared to the doubly-phosphorylated β-catenin peptide -based binding assay utilizing β-catenin phosphodegron peptides (residues 17–48) and recombinant β-TrCP/Skp1 complex (referred as β-TrCP subsequently). Consistent with the role of the phosphodegron in targeting β-catenin to β-TrCP, the doubly-phosphorylated peptide (pSer33/pSer37) binds with 2 nM binding affinity whereas the non-phosphorylated peptide (Ser33/Ser37) binds with significantly lower affinity (>100 μM) Fig. . Removal30  of 206 ± 54 μM, as well as in orthogonal TR-FRET and SPR binding assays with similar EC50 values of 246 ± 17 μM and 129 ± 33 μM, respectively  and functions as a phosphate mimetic that occupies the β-catenin Ser37 phosphate binding site and mimics the phosphate by forming a hydrogen bond with the backbone N–H of Gly432 on β-TrCP. Lastly, the phenyltetrazole tail group extends away from the β-catenin:β-TrCP interface, with the phenyl ring stacking against Arg431 of β-TrCP and the anionic tetrazole residing in close proximity to the cationic Arg410 and Arg431 residues of β-TrCP , bound to the β-catenin:β-TrCP complex Fig. . The strrCP Fig. .Fig. 2NRβ-TrCP. Consistent with the defect in β-TrCP binding, the pSer33/Ser37 β-catenin peptide displayed reduced ubiquitylation relative to the doubly-phosphorylated peptide. Importantly, NRX-1532 weakly enhanced the ability of β-TrCP to ubiquitylate the pSer33/Ser37 β-catenin peptide , we monitored the binding affinity of β-catenin to the β-TrCP at increasing concentrations of compound Fig. . In a TR31, the cationic nature of the phosphate binding site, and the increased entropic penalty of forming a ternary complex. However, analysis of the bound NRX-1933 crystal structure b-factors indicates that the N-terminus of the β-catenin peptide has the potential for movement. Therefore, we initially focused on strategies to induce movement in this region of β-catenin to induce a larger, more druggable binding pocket. This strategy was realized early in the design campaign, with analogues such as NRX-2663 (3), where phenoxy substitution at the 4-position of the trifluoromethyl pyridone core induces rearrangement of β-catenin Leu31 to create a larger binding pocket  and cooperativity of binding  with pSer33/Ser37 β-catenin peptide . Computational docking of NRX-252114 bound to the β-catenin:β-TrCP complex suggested that the cyanoisoindoline projects out of the plane from the trifluoromethylpyridone and makes additional hydrophobic contacts with β-TrCP. NRX-252114 enhances the binding of pSer33/S37A β-catenin peptide for β-TrCP with >1500-fold cooperativity, surpassing the affinity of WT doubly-phosphorylated peptide . Compared to the original HTS hit NRX-1532, with an EC50 > 200 μM and only eight-fold cooperativity, NRX-252114 and NRX-252262 represent significant improvements in both measures. Importantly, improvements in both potency and cooperativity translated into enhanced ubiquitylation of mutant β-catenin peptide, with enhancer molecules potentiating ubiquitin chain formation to levels greater than WT β-catenin peptide.The small molecule enhancers NRX-1532 and NRX-1933 have reasonable molecular weight and drug-like physiochemical properties to serve as starting points for a structure-enabled design campaign to improve both potency and cooperativity ; however, in the presence of 20 μM NRX-252114 that binding affinity is enhanced to 180 nM  and potency  of the enhancers. This required improving affinity by increasing ligand efficiency, rather than improving affinity by increasing ligand molecular weight and growing away from the PPI interface. This allowed the enhancers to retain drug-like physiochemical properties throughout the optimization process even though they evolved from a weak screening hit.Here we report the prospective discovery and rational design of small molecule molecular glues that enhance a substrate:ligase interaction to induce substrate degradation. These molecules potentiate the binding of S37A mutant β-catenin to β-TrCP. The enhanced PPI affinity results in increased K48-linked ubiquitylation of mutant β-catenin by its natural ubiquitin ligase SCF34 this work represents a prospective discovery of such molecules for a specific ligase-substrate pair. Compared to the better characterized enhancers, auxin and the IMiDs that directly bind to their respective ligases to create surfaces for substrate binding10, these enhancers do not exhibit appreciable binding directly to β-TrCP alone and only bind in the context of the ternary complex. The enhancers herein also differ from the other known substrate:ligase enhancers by promoting the interaction of β-TrCP with its native substrate, β-catenin, that has otherwise lost affinity because of its mutational status. The enhancer molecules provide the essential binding surface between β-catenin and β-TrCP that is lost as a result of the S37A β-catenin mutation. In contrast, the IMiD enhancers promote the binding of neo-substrates IKZF1, IKZF3, CK1α, and GSPT1 to the ubiquitin ligase CRBN32, while another enhancer, indisulam, promotes the binding of neo-substrate Rbm39 to the ubiquitin ligase DCAF1534. These previously reported substrates have no endogenous connection to the ligase and display no binding affinity in the absence of enhancers. Compared to hetero-bifunctional molecules, these enhancers have an acceptable molecular weight for small molecules with desirable drug-like properties. Rather than degrading substrates with bifunctional molecules by hijacking an unrelated ligase where tissue-specific expression of both proteins could be a challenge to drive ternary complex formation for efficient ubiquitylation, these enhancers co-opt the native ligase to degrade substrate proteins that has otherwise lost binding due to mutations. Additionally, utilizing a native substrate-ligase pair that enhances PPI through the naturally occurring interface ensures both cooperative interaction as well as provide accessible substrate lysines for efficient ubiquitylation.While a few retrospectively characterized enhancers of substrate:ligase interactions have been reported27. Our work provides an approach to target the Wnt signaling pathway by promoting degradation of mutant β-catenin by SCFβ-TrCP. Since these enhancer ligands bind at the phosphoserine binding site on β-TrCP, binding of the doubly-phosphorylated WT β-catenin is not enhanced by these ligands. This ensures the specificity of the enhancers for the mutant form of β-catenin without impacting the function of WT β-catenin in normal tissues. Due to the uncertainty of β-catenin Ser33 phosphorylation status in the mutant TOV-112D cells, we utilized an engineered Ser33 phosphomimetic β-catenin model to demonstrate cellular mechanistic proof-of-concept for mutant β-catenin degradation. Additionally, the small molecule NRX-252114 enhanced the affinity of unphosphorylated Ser33/S37A β-catenin for β-TrCP from >10,000-fold to within 90-fold of WT doubly-phosphorylated β-catenin affinity. Importantly, these enhancers potentiated unphosphorylated β-catenin ubiquitylation with chain lengths similar to that of doubly-phosphorylated WT β-catenin. This tantalizing result suggests that further potency improvements to the small molecule could drive unphosphorylated β-catenin ternary complex formation in cellular and in vivo contexts, and enable degradation of unphosphorylated β-catenin in β-catenin dependent tumors typically observed in cancers with APC, Axin or β-catenin mutations. This work shows that a prospective discovery approach can be rationally applied to develop small molecule β-catenin degraders beyond Ser37 mutations.Developing small molecule therapeutics targeting the Wnt/β-catenin signaling pathway remains a challenge despite extensive efforts to target various components of the pathway28. Dysregulation often results from mutations leading to compromised binding of transcription factors to their cognate ligases. This work provides a rational framework to prospectively employ small molecules that target such oncogenic transcription factors by enhancing substrate:ligase interactions to promote their degradation. Ultimately, this strategy could be useful to target therapeutically important classes of proteins that were previously deemed undruggable and might find further application to other PPIs of different classes as well.Transcription factors such as β-catenin are implicated in the development of many diseases, but with few exceptions, effectively remain as an undruggable target class. It is now evident that post-translational modification via the ubiquitin proteasome system is a common mechanism by which the levels of transcription factors are regulated in cells30 were cloned and co-expressed in E. coli in pCDF-Duet1 vector as 6xHis-SMT3-β-TrCP1/Skp1 (for FP) or as 6xHis-SMT3-β-TrCP1-avi tag/Skp1 . Cultures were grown at 37 °C to an OD600 of 1.0 and then induced for 16 h with 1 mM IPTG at 18 °C. 0.5 mM d-biotin was also added to cultures containing avi-tagged β-TrCP and BirA at the time of induction. Bacteria were pelleted and lysed in buffer containing 50 mM NaH2PO4 pH 8.0, 500 mM NaCl, 20 mM Imidazole, 5 mM BME, and protease inhibitors . The lysate was incubated with Ni-NTA agarose (Qiagen), washed, and eluted with buffer containing 250 mM imidazole. Eluate was cleaved by ULP1, buffer exchanged overnight, and re-purified by Ni-NTA agarose to remove the cleaved 6xHis-SMT3 as well as any un-cleaved protein. The unbound fraction was further buffer exchanged into low salt, low pH buffer , bound to a 5 mL HiTrap SP column (GE) and eluted by a linear salt gradient. Fractions containing β-TrCP/Skp1 complex were concentrated and further purified by size exclusion chromatography   was over-expressed in HEK293T cells for 48 h following transfection using FuGeneHD. In some instances, to increase yield, cells were treated with 5 μM Nedd8 E1 inhibitor (MLN4924) for 4 h prior to harvest. Cells were lysed in 50 mM Tris Cl (pH 7.5), 150 mM NaCl, 5% glycerol, 1% NP-40, protease and phosphatase inhibitors and bound to FLAG resin (Sigma), washed and eluted with 150 μg/ml 3× FLAG peptide. The eluted protein was concentrated, FLAG peptide was removed using spin column and analyzed , E2 (6xHis-TEV-Cdc34a), and Cul1-Rbx1 (6xHis-TEV-Cul1 and GST-TEV-Rbx1) were expressed in either Y = Bmin + (Bmax − Bmin)(X^h)/(X^h + EC50^h)) in Prism (GraphPad). Errors are standard error of the fit.Compounds were incubated in a 20 μl reaction volume with β-TrCP/Skp1 (116 nM in binding enhancement assays and a concentration dilution series in peptide binding and cooperativity assays) and 5 nM BODIPY-TMR labeled β-catenin peptides (see figure legends for peptide details) in a black 384-well microplate (Perkin Elmer ProxiPlate), in a PBS buffer containing 0.01% Triton X-100 and 2 mM DTT with 2% final DMSO. Following 1-h incubation at room temperature, plates were read on an Envision plate reader (Perkin Elmer) for FP signal using a P-pol and S-pol 595/60 filter and a D555 single mirror. Binding and enhancement data were fit to a standard Hill equation . TR-FRET signal was calculated using a 520/620 nm ratio. Binding and enhancement data were fit to a standard Hill equation (Y = Bmin + (Bmax − Bmin)(X^h)/(X^h + EC50^h)) in Prism (GraphPad). Errors are standard error of the fit.Compounds were incubated in a 20 μl reaction volume with 300 pM biotinylated β-TrCP/Skp1 preincubated with 1.2 nM terbium-coupled streptavidin (Cisbio) and BODIPY-FL labeled β-catenin peptides (see figure legends for peptide details) in a white 384-well microplate (Perkin Elmer ProxiPlate), using a PBS buffer containing 0.01% Triton X-100 and 2 mM DTT with 2% final DMSO. In binding enhancement assays, the peptide concentrations were held constant at 107, 85, 10, 5, and 85 nM for the pSer33/Ser37, pS33/S37A, pS33/S37C, pS33/S37F, and S33E/S37A peptides, respectively. In peptide binding and cooperativity assays, the peptide concentrations were varied across the concentration range displayed on the High-throughput screen activity was assessed with FP (as described above) and calculated using the percent change from the null control and standard deviation (σ) from the plate median. Hit thresholds were defined by two standard deviations from the plate median or 25% enhancement from the null control.Y = Bmin + (Bmax − Bmin)(X^h)/(X^h + EC50^h)) in Prism (GraphPad). Errors are standard error of the fit.SPR binding assays were performed on a GE Healthcare Biacore T200 using a Series B Biacore Sensor Chip SA. Biotinylated β-catenin pSer33/Ser37 peptide was immobilized at 27.6 RU. 333 nM β-TrCP/Skp1 and titrated compound were preincubated and flowed at a rate of 30 μl/min in 10 mM HEPES, 150 mM NaCl, 0.05% Surfactant P20, 1 mM TCEP, and 2% DMSO at pH 7.4 and 20 °C. Engagement of the ternary complex was assessed with a contact and dissociation time of 120 s. Equilibrium binding levels were assessed using Biacore T200 Evaluation Software (GE) and these data were fit to a standard Hill equation  peptide were mixed one-to-one with a well solution of 0.1 M HEPES pH 7, 10% PEG 6000 and incubated over that well solution at 20 °C. Three to four crystals from this condition were pulverized in 100 µL of well solution using the Hampton Research Seed Bead kit and further diluted 1:1000 with well solution. Subsequent crystals were obtained by adding 0.2 µL of this diluted seed stock to a 2 µL drop containing 1 µL of 8.2 mg/mL β-TrCP/Skp1 and a two-fold molar excess of either pSer33/Ser37 β-catenin peptide  or pSer33/S37A β-catenin peptide (Ac-CDRKAAVSHWQQQSYLD(pS)GIHAGATTTAPSLSG; NRX-103094 containing crystal), and 1 µL of a well solution containing 8% PEG4000, 100 mM BTP pH 5.5. After growth, crystals were transferred to a 2 µL drop of soaking solution containing 10% PEG 4 K, 0.1 M HEPES pH 7.1, 2 mM DTT, 400 µM β-catenin peptide, 10% DMSO and 1–10 mM of compound. These drops were suspended over a well of 10% PEG4000, 0.1 M HEPES pH 7.1 overnight. Crystals were cryoprotected in soaking solution supplemented with 18% ethylene glycol and frozen in liquid nitrogen.37. For the highest resolution NRX-2776 containing structure, the coordinates of β-TrCP and Skp1 from PDB ID 1P2230 were used as search ensembles in PHASER38 to obtain an initial solution for this structure by molecular replacement. The final model was built through iterative rounds of refinement in Phenix40 and Coot41. Ligand restraints were generated in Maestro and Phenix Elbow43. The resulting model for the NRX-2776 containing complex was used as the starting point and refinement constraints for refinement of the other structures , 95.5/4.3/0.2 (NRX-2663), 95.5/4.3/0.2 (NRX-103094), 95.7/4.4/0.0 (NRX-1933), and 95.5/4.5/0.0 (pSer33/Ser37) percent Ramachandran favored/allowed/outliers. Figures were made using PyMol45.Data for the pSer33/Ser37, NRX-2663, NRX-1933, and NRX-103094 containing crystals were collected at the Canadian lightsource on beamline 08ID at 100 K using and a wavelength of 0.9795 Å. Data for the NRX-2776 containing crystal were collected at the Advanced Photon Source on beamline 22ID at 100 K using and a wavelength of 1 Å. Data were processed using XDS and scaled in XSCALE, using CC1/2 as the resolution cutoff criteriaThe pKa values were determined using the fast UV (spectrometric) technique. Samples were titrated in triplicate under methanol:water co-solvent conditions (with MeOH:water mixing ratios varied from 1:1 to 1:4) in the pH range 2.0–12.0 at concentrations of 15 to 30 μM and at a temperature of 25 °C. Aqueous pKa values were determined by Yasuda-Shedlovsky extrapolation from the individual determined results.The X-ray crystal structure of NRX-103094 was used in the docking of the various analogs. The structure was prepared with the protein preparation procedure in Maestro. All hydrogens were added, and charge states defined using PROTKA procedure, follow by h-bond optimization and constraint relaxation to 0.2 rmsd tolerance. The resulting structure was used to generate docking grids and perform docking studies using GLIDE. The GLIDE extra precision scoring function (XP) was used to obtain suitable docking poses, which were subsequently refined with Macromodel embrace procedure using default options. All the modeling was done with Maestro 10.3 Release 2015-2 (Schrodinger LLC).2, 2 mM ATP, and 1 mM DTT at room temperature in the presence of 62.5 μM Ubiquitin, 125 nM Ube1, 1.75 μM Cdc34A, and 100 nM neddylated SCFβ-TrCP in the presence or absence of compound (as indicated). Typically, individual reaction mixes with either Ubiquitin, Ube1 and Cdc34A or β-catenin, SCFβ-TrCP and compound were preincubated and subsequently mixed to initiate β-catenin ubiquitylation. Reactions were quenched by SDS loading buffer, resolved by SDS-PAGE and imaged for fluorescence. For full length β-catenin protein ubiquitylation 400 nM β-catenin and 400 nM SCFβ-TrCP were used instead.Four micromolar of fluorescently-labeled β-catenin peptide (residues 17–60) were ubiquitylated in a buffer containing 25 mM Tris Cl (pH 7.5), 50 mM NaCl, 5 mM MgCl6 cells. Lysates were clarified by centrifugation at 20,000 × g for 20 min at 4 °C and the supernatant was collected for subsequent use. Purified Myc/FLAG-tagged wild-type β-catenin protein at 2 µM was pretreated with 1 µM GSK3β , 50 nM Axin , 200 nM CK1α , and 2 mM ATP at 30 °C for 60 min. This phosphorylated β-catenin, unphosphorylated WT β-catenin, or S33E/S37A β-catenin were each diluted into lysate to 100 nM, at the indicated compound concentrations, at a final DMSO concentration of 1% DMSO. Thirty-five microliters of these mixtures were incubated over 10 µL of anti-c-myc magnetic beads , with rotation, at room temperature for 2 h. Beads were then pelleted, washed twice with 200 µL cold wash buffer  containing compound at the same incubation concentration. Washed beads were resuspended in 30 µL SDS loading dye and analyzed by western analysis.Lysate pulldown with recombinant β-catenin: HEK293T cells were lysed by freeze thaw in lysis buffer , phosphatase inhibitors , 5 nM Calyculin A ) at 85 µL of buffer to 1 × 106 cells per plate. Cells were transfected 24 h later with 2.5 µg of a WT or S33E/S37A Myc/FLAG-tagged β-catenin expression construct using the transfection reagent FuGeneHD. Forty-eight hours post transfection, cells were treated with a combination of 20 µM MG132 and NRX-252114 at the described concentrations. Cells were then liberated from 10 cm dishes using a cell scraper, pelleted, washed with PBS, resuspended in an equal volume of hypotonic lysis buffer , and sheered by eight passages through 21 G and 25 G needles. Samples were then supplemented up to 150 mM NaCl and clarified by centrifugation. Thirty microliters of each lysate was incubated over 10 µL of anti-c-myc magnetic beads  and incubated at room temp with rotation for 30 min. Beads were pelleted, washed twice with lysis buffer, and resuspended in SDS loading buffer. Samples were analyzed by western blot.Pull down from cells treated with compound: HEK293T cells were plated in 10 cm dishes at a density of 1.2 × 10To monitor ubiquitylated β-catenin in lysates, treated cells (as indicated in figure) were lysed in lysis buffer  containing proteases inhibitors , phosphatase inhibitors , 5 nM Calyculin A , 20 µM MG132 , 10 mM NEM , 5 mM IAA , 2 mM OPT  and 50 µM PR-619 . Clarified lysates were resolved by SDS-PAGE followed by western analysis.HEK293T cells were plated in a 10 cm dish in regular growth media (DMEM + 10% FBS) and allowed to adhere to the plate overnight. The following day, cells were transfected with S33E/S37 β-catenin using FuGeneHD transfection reagent. Transfected cells were then selected with media containing 700 μg/ml Geneticin. A pooled population of selected cells were used for all described experiments.5 cells/well. The following day, cells were incubated with compounds diluted in regular growth media to the appropriate concentration. The final concentration of DMSO for all cell treatments was kept at 0.5% or less. Cell treatments with the Nedd8 inhibitor (MLN4924) and the proteasome inhibitor (MG132) were performed at a final concentration of 5 μM and 10 μM respectively. Cycloheximide treatment was performed at a final concentration of 100 μg/ml.HEK293T cells expressing S33E/S37A β-catenin were plated in six-well plates at a density of 9 × 105 cells/well. The following day, cells were transfected with a non-targeting siRNA or a combination of Dharmacon SMARTPool siRNAs targeting β-TrCP1 & β-TrCP2 using Dharmafect1 lipid reagent (Dharmacon/Horizon Discovery). Transfected cells were grown for 48 h before being re-plated in six-well plates at a density of 9 × 105 per well. The following day (72-h post siRNA transfection), cells were treated with either DMSO or NRX-252114 for 6 h. Harvested lysate was assessed for levels of FLAG-tagged S33E/S37A β-catenin by Western analysis.HEK293T cells expressing S33E/S37A β-catenin were plated in six-well plates at a density of 5 × 10TOV-112D cells were plated in six-well plates followed by infection with β-catenin shRNA lentivirus . Infected cells were then selected with blasticidin for three days before being used for subsequent experiments. Effects of β-catenin knock-down on cell proliferation were monitored using CelltiterGlo (Promega) reagent under standard conditions.HEK293T or TOV-112D cells were treated with 20 μM MG132 for 4 h with or without 5 nM Calyculin A for 40 min. Cells were lysed in the presence of protease and phosphatase inhibitors, run on SDS-PAGE and analyzed by western analysis. The ubiquitylated β-catenin species were monitored using either anti-pS33 β-catenin antibody or anti-pS33/37 β-catenin antibody.Details of chemical syntheses can be found in the Supplementary Information.β-Catenin (17–48): Ac-CDRKAAVSHWQQQSYLDpSGIHSGATTTAPSLSGβ-Catenin (17–60): Ac-CDRKAAVSHWQQQSYLDpSGIHSGATTTAPSLSGKGNPEEEDVDTSIƙBα: Ac-PRDGLKKERLLDDRHDpSGLDSMKDEEYEQMVKELQEIRLEPQEVWee1: Ac-CEEEEEEEGSGHSTGEDpSAFQEPDSPLPPARSPTEPGPERRRSPGEmi1: Ac-CSTNEIEALETSRLYEDpSGYSSFSLQSGLSEHEEGSLLEENFGDSCell lines were obtained from the American Type Culture Collection: HEK293T (ATCC- CRL-3216) TOV-112D (ATCC- CRL-11731).Antibodies purchased from Cell Signaling Technologies: Anti-pS33/37 β-catenin #2009S (1:1000); Anti-pT41/S45 β-catenin #9565S (1:1000); Anti-β-catenin (C-ter) #9587S (1:2000); Anti-FLAG #2368 (1:2500); Anti-β-TrCP #4394 (1:1000). Anti-Actin was from Sigma, Catalog #A5441 , and Anti-pS33 β-catenin from GeneTex, Catalog #GTX50255 (1:1000)Further information on experimental design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


AbstractThis paper presents an updated list of tracheophytes of Xiao Hinggan Ling. The list includes 124 families, 503 genera and 1640 species (Containing subspecific units), of which 569 species (Containing subspecific units), 56 genera and 6 families represent first published records for Xiao Hinggan Ling. The aim of the present study is to document an updated checklist by reviewing the existing literature, browsing the website of National Specimen Information Infrastructure and additional data obtained in our research over the past ten years.This paper presents an updated list of tracheophytes of Xiao Hinggan Ling. The list includes 124 families, 503 genera and 1640 species (Containing subspecific units), of which 569 species (Containing subspecific units), 56 genera and 6 families represent first published records for Xiao Hinggan Ling. The aim of the present study is to document an updated checklist by reviewing the existing literature, browsing the website of National Specimen Information Infrastructure and additional data obtained in our research over the past ten years.This is the first complete checklist of tracheophytes of Xiao Hinggan Ling. A total of 1640 species and subspecies are listed, of which 569 species (ontaining subspecific units) represent first published records. Pinuskoraiensis, known as "The Home Of Korean Pine"  includes 1161 species belonging to 439 genera and 114 families . Among thttp://www.ipni.org/index.html) and Global Biodiversity Information Facility (GBIF) (http://www.theplantlist.org/1.1/about) by Taxonomic Name Resolution Service (TNRS) (http://tnrs.iplantcollaborative.org). Finally, we unified the classification system. PPGI system was used for Pteridophyte , “Floristics and distribution of plants in Xiao Hinggan Ling, China” , The Inty (GBIF) , the newy (GBIF) , and Floy (GBIF) , Speciesy (GBIF) . Scientiidophyte , Christeidophyte , and APGidophyte  (Suppl. MildeL.(L.) KuhnDesf.L.Kom.(Jacq.) Woyn. ex Schinz & Thell.(L.) All.Maxim.(Mayr) Pilg.Shiras.Ledeb.Franch.Siebold & Zucc.L.Lej.L.L.All.(Makino) H.HaraMiq.L.Pourr.L.L.(Jacq.) Fisch. & C.A.Mey.Shan et YuanMakino & Jabe(Kitag.) Y. Huei HuangHoffm.Schischk.(Makino ex Y.Yabe) Nakai(Miq.) Franch. & Sav.Nakai(Kitag.) PimenovMaxim.(L.) Hoffm.(Franch.) Kitag.HelmDunnDielsMichx.(Diels) F.T. Pu(Makino ex K. Yabe) MakinoMaxim.Kitag.Wall.L.Ledeb.Turcz. ex Ledeb.RegelMaxim.L.G.DonFisch. ex LinkBungeL.(Miq.) Ohwi(C.Morren & Decne.) H.HaraMaxim.Baroni(L.) L.Pall. ex LinkPall.Schltr.(L.) CrantzS. C. ChenMaxim.(Maxim.) NakaiNakai(Kom.) Y.L.Chen(Ledeb.) Y.L.Chen(Thunb.) KrylovH.Lév. & VaniotDC.HerderNakai & Kitag.C.B.ClarkeKitag.Kom.Maxim.Willd.Turcz. ex DC.(Koidz.) Kitam.Nakai & Kitag.TauschIljinLour.NevskiDahlst.Hand.-Mazz.DielsKitag.(Turcz. ex DC.) Holub(L.) Rchb.(Nakai) C.Jeffrey & Y.L.Chen(Maxim.) Pobed.(Nannf.) D.Y.Hong & S.GeNakaiKom.Nakai(Ledeb.) Kitag.Hemsl.(Hornem. ex Spreng.) Domin PopovKitag.(Schultz) Hyl. ex Nordh.(Nakai & Kitag.) W.T. Wang(Fisch. ex DC.) O'Kane & Al-Shehbaz(L.) O'Kane & Al-ShehbazDC.With.NakaiL.L.Maxim.C.A.Mey.Fisch. & C.A.Mey.(Georgi) BobrovWilld.Schrad.Bunge ex Maxim.Bunge ex Maxim.L.L.(L.) Mosyakin & Clemants(Fisch. ex Ser.) F.N. WilliamsFenzlL.Willd.L.(Poir.) FenzlMiq.(Regel) Maxim.(Nakai) Hara(Turcz.) Pax(L.) RothMaxim.Willd.(Robill. & Castagne ex DC.) Benth.L.(Bunge) KuntzeKitag.(L.) K.Koch(F.Schmidt) HolubGray(L.) Ronse Decr.(Kom.) A.J.Li(Lange) Small(Makino) Kitam.Makino(Roxb.) StewardRegelMeisn.L.M.Bieb.Buch.-Ham. ex D. DonSkvortsovLaxm.(Makino) Kudô & Masam.L.L.Rchb.L.Ledeb.Thunb.Wall.Miq.Loes.Ledeb.L.Juz.L.L.(Siebold & Zucc.) HarmsDielsBungeL.NakaiNakai RehderL.Franch.Thunb.L.NakaiBungeLinkHemsl.L.L.DubyL.(L.) K.Richt.E.A. BuschMaxim.Kom.BlumeDecne.Maxim.Rupr.Regel Pers.Maxim.Turcz.(DC.) X.F. GaoMedik.Kitag.L.f.Bunge(DC.) Poir.Pall.G.SimpsonLam.Oliv.(L.) Merr.(Miq.) Litv.H.Lev.Miq.BungeL.L.L.Ledeb.Hosok.(Link) Maxim.RothThunb.MayrBunge(Willd.) Rech.f.T.N.Ho(Miq.) Maxim. ex Franch. & Sav.Fawc.L.(Maxim.) Pobed.L.L.Kom.(Y.L.Chang) C.C.Huang & L.R.XuDC.Kom.Franch. & Sav.Serg.Maxim.(Turcz.) Kamelin & Makhm.(Regel) Kamelin & Makhm.(Benth.) KuntzeL.L.Benth.Nakai ex F.Maek.(Thunb.) Kudô(Siebold & Zucc.) MennemaMaxim.L.L.RegelKom.Maxim.(Juz.) C.Y.Wu & H.W.LiHemsl.(Regel) H.Hara(Miq.) H.Hara(Krock.) Philcox M.C.Chang & X.L.ChenFreyn(Freyn) Hong(Bunge) TsoongL.(Koidz.) HondaOliv.L.Mill.(Ganesch.) Tzvelev(Nakai & Kitag.) D.Y. HongStevenNakai(L.) WaltersPall. ex Link(Turcz. ex Trautv.) Ledeb.(Ledeb.) Schult. & Schult.f.BakerKom.Salisb.Miq.A.DC.(Rehder & E.H.Wilson) Nakai(L.) Nied.(D.Don) H.HaraKlenze ex Alef.Maxim.L.Y.L. Chou & Y.L. ChangTurcz.Turcz.Boiss.Steud.L.L.Rupr.L.C.Morren & Decne.L.Rupr. & Maxim.L.CarrièreSarg.Pall.K.S.HaoTurcz.SeemenTrautv.Willd.RusbyL.Cav.BungeHemsl.(Roxb.) MakinoL.(Bunge) Hand.-Mazz.Maxim.Scop.Thunb.L.Hausskn.MosquinRaf.Hausskn.Salisb.Burm.f.Edgew. & Hook. f.L.LandoltLandoltKom.(Vahl) Schinz & Thell.(F.Schmidt) T.V.Egorova(Ohwi) Y.C.Yang & M.ZhanR.Br.(H.Lév. & Vaniot) S.Yun Liang, L.K.Dai & Y.C.TangOhwiKom.Kom.OhwiKom.OhwiKük.Turcz.Maxim.BoottKük.C.A.Mey. ex Meinsh.H.Lév. ex T.KoyamaKom.Maxim.Steud.(L.) Roem. & Schult.(L.) Roem. & Schult.Zinserl.(Roem. & Schult.) T.KoyamaVahlMakino(Hochst. ex Steud.) Karthik.(Roxb.) LyeOhwiL.NakaiMaxim.L.BuchenauA.DC.Roxb.(Hance) A.E.MurrayL.Keng f.(Thunb.) MakinoL.KengL.MezPhil.Maxim.Lam.(Huds.) P.Beauv.LinkGriseb.(Ard.) Fritsch(Ohwi) S.L. Chen & G.H. Zhu(Ohwi) S. L. ChenHostMakinoL.Steud.(Gorski) GorskiL.Griseb.(L.) R.Br.Steud.OhwiL.(H.Lév.) C.D.K.Cook & M.S.NichollsFunckSkvortsovL.(Thunb.) Less. ex Less.Sch.Bip.Kom.Franch.Kitam.HayataWilld.VaniotKitag.Maxim.Wall. ex BesserY.R.LingL.Thunb.(Nakai) Kitam.(Thunb.) DC.(Thunb.) DC.L.Willd.L.Franch. & Sav.L.Ramat.HerbichPetr.(Maxim.) C.Shih(L.) Pers.L.A.GrayDC.(Vant.) WangMaxim.Komar.(Kom.) Kom.L.(Turcz.) B.Nord.NakaiNakaiSiebold & Zucc.Mandl & MuhldorfMaxim. ex Regel(Y.H.Chou & Chun C.Hsu) W.T.Wang ex Z.Y.Su & C.Y.WuMaxim.(Clerc) Krecz.(L.) Rchb.Kom.(Rchb.) RegelNakaiNakaiNakaiKoelleS.H.Li & Y.H.HuangFranch.Kitag.(Nakai) OhwiRegelMaxim.C.A.Mey.Siebold & Zucc.(Willd.) Trevir.Kitag.L.Turcz.DC.BungeLedeb.(Maxim. ex Kuntze) Hand.-Mazz.NakaiMaxim.RehderKrylov & Serg.Maxim.(Kitag.) W.T. WangOvcz.Ovcz.(Laest.) C.D.K.CookMaxim.L.L.L.(Ledeb.) RegelL.Miq.Rchb.Maxim.NakaiBungeL.L.Bunge(L.) Kurtto & T.Erikss.NakaiWilld.(Maxim.) Kom. ex Juz.L.(Franch. & Sav.) Koidz.Maxim.Willd. ex Schltdl.Wight & Arn.H.Lâ€šv.Th.WolfL.Peterm.L.BungeRegel & TilingLindl.Lindl.(C.K.Schneid.) KoehneL.BungeC.A. MeyerDieck ex Focke(Michx.) FockeL.L.(Kitag.) T.T.Yu & C.L.LiMaxim.Maxim.(Koidz.) Rehder(Rupr.) Maxim.Turcz.NakaiPlanch.Jacq.L.G.Forst.(Wedd.) FriisL.C.A.Mey.L.Bunge(Boreau) H. Ohba(Y.C. Chu ex S.H. Fu & Y. Hui Huang) H. Ohba, K.T. Fu & B.M. Barthol.Raym.-HametFisch.Freyn(L.) Scop.L.F. SchmidtJancz.Pojark.Kom.L.Pall.Jancz.L.(Hemsl.) Engl.NakaiKitag.OhwiHand.-Mazz.Nakai & Takeda(Engl.) Kom.CurtisBrummittLam.L.(Thunb.) Momiy.(Maxim.) Momiy.L.Acoraceae, Butomaceae, Linderniaceae, Moraceae, Pedaliaceae and Zygophyllaceae and the results of ranking 56 newly added genera by genus size are shown in Table A total of 124 families, 503 species and 1640 species of tracheophytes in Xiao Hinggan Ling have been compiled. Compared with the list of “Floristics and Distribution of Plants in Xiao Hinggan Ling China”, The updated list adds 569 species, 56 genera and 6 families to the old list  . Second, some new record species were found through our field survey, such as some invasive species and exotic plants (e.g.annuus) . The pheannuus) .The new list can reflect the plant varieties in the natural ecosystem of Xiao Hinggan Ling. It can provide detailed and reliable basic data for the study of flora, biodiversity and alien plant invasion in Xiao Hinggan Ling .Supplementary material 1New Checklist of vascular plants in Xiao Hinggan Ling-completeData type: ChecklistFile: oo_248961.xlsHongfeng Wang


Circular RNAs are a class of regulatory RNA transcripts, which are ubiquitously expressed in eukaryotes. In the current study, we evaluate the function of a novel circRNA derived from the β-catenin gene locus, circβ-catenin.Circβ-catenin is predominantly localized in the cytoplasm and displays resistance to RNase-R treatment. We find that circβ-catenin is highly expressed in liver cancer tissues when compared to adjacent normal tissues. Silencing of circβ-catenin significantly suppresses malignant phenotypes in vitro and in vivo, and knockdown of this circRNA reduces the protein level of β-catenin without affecting its mRNA level. We show that circβ-catenin affects a wide spectrum of Wnt pathway-related genes, and furthermore, circβ-catenin produces a novel 370-amino acid β-catenin isoform that uses the start codon as the linear β-catenin mRNA transcript and translation is terminated at a new stop codon created by circularization. We find that this novel isoform can stabilize full-length β-catenin by antagonizing GSK3β-induced β-catenin phosphorylation and degradation, leading to activation of the Wnt pathway.Our findings illustrate a non-canonical function of circRNA in modulating liver cancer cell growth through the Wnt pathway, which can provide novel mechanistic insights into the underlying mechanisms of hepatocellular carcinoma.The online version of this article (10.1186/s13059-019-1685-4) contains supplementary material, which is available to authorized users. As a global health problem, liver cancer is a devastating disease with high mortality rate. The known etiologies for liver cancer include chronic virus infection, cirrhosis, long-term alcohol abuse, hepatic steatosis, and aflatoxin exposure . The higIn the past decades, numerous signaling pathways have been identified to get involved in hepatocarcinogenesis, including Notch pathway, PI3K/Akt pathway, and Wnt/β-catenin pathway . As a hiThe regulatory RNA world includes a wide spectrum of RNA molecules such as microRNA, Piwi-interacting RNA, small interfering RNA, and long non-coding RNA. With the recent advances in high-throughput sequencing techniques and bioinformatics tools, numerous circular RNAs (circRNAs) have been identified in various human tissues and cell lines , 13. TheIn this study, we analyzed the RNA sequencing data from an online database and identified a circRNA derived from the β-catenin gene, which was named “circβ-catenin.” Functional investigations showed that knockdown of circβ-catenin suppressed in vivo and in vitro cancer cell growth. Subsequent studies displayed that circβ-catenin encoded a novel 370-amino acid β-catenin isoform. This isoform protected β-catenin from GSK3β-mediated degradation and thus potentiated the activation of Wnt/β-catenin pathway.In terms of the pathological importance of Wnt/β-catenin pathway in hepatocarcinogenesis, we selected 143 Wnt pathway-related genes and examined how many circular RNAs were derived from these genes by analyzing the circular RNA sequencing data from online database circBase  β-catenin is a master regulator of Wnt pathway in liver cancer, 2) this circRNA was the only circular RNA which was widely detected in three independent sequencing datasets  was 1129 nucleotides and contained six exons. By using a divergent primer in cDNA samples and Sanger sequencing, we confirmed that the head-to-tail splicing (back-splicing) occurred in the exons from β-catenin  sequence and it might encode a novel β-catenin isoform with 370 amino acids Fig. b. To conNext, we inserted the full-length sequence of β-catenin-370aa into a plasmid with HA tag Fig. e. The imBecause silence of circβ-catenin suppressed the protein level of β-catenin without affecting the RNA level of β-catenin, we thus speculated that circβ-catenin might modulate the expression of β-catenin in protein level rather than the transcription level. Previous studies demonstrate that the stability of β-catenin is tightly linked with its phosphorylation status. Upon phosphorylation by GSK3β at three residues (Ser33/Ser37/Thr41), β-catenin is ubiquitinated by ubiquitin ligase β-TrCP and subsequently degraded by the proteasome. Therefore, we examined the phosphorylation status of β-catenin. We found that silencing of circβ-catenin decreased the phosphorylation at Ser33/Ser37/Thr41 sites Fig. a. In terWe next evaluated the effect of circβ-catenin in modulating the mutual interaction between GSK3β and full-length β-catenin. Based on the co-IP results, we found that silencing of circβ-catenin enhanced the interaction between GSK3β and full-length β-catenin Fig. c whereasIn the past decades, numerous investigations have displayed abnormal expression profiles of non-coding RNAs in a wide spectrum of cancer types. Recent studies have unraveled that many miRNAs and lncRNAs played vital roles in modulating tumor growth and cancer metastasis , 5. NoneIn the current study, we find that β-catenin-370aa serves as a decoy and competitively prevents binding of GSK3β to full-length β-catenin protein. According to the results from western blot, we find that β-catenin-370aa is around 3-fold less abundant than the full-length β-catenin protein Fig. d. We alsAlternative splicing of linear mRNA provides the potential to generate a large number of diverse protein isoforms from limited protein-coding genes within the human genome . IntriguIt was recently reported that artificial circRNA can be translated independently of the IRES sequence , 30. EmeIn conclusion, herein, we identify a novel circRNA derived from β-catenin, a well-characterized oncogene in liver cancer. This is the first report that circRNA can encode a novel protein isoform, which subsequently promotes tumor growth through activation of the Wnt/β-catenin pathway in liver cancer. These findings expand our current knowledge of circRNAs and indicate that the protein-coding capacity of non-coding RNAs especially circRNAs is largely underestimated.−ΔΔCT method. The primer sequences were provided in Additional file The total RNA was isolated using the FavorPrep™ Tissue Total RNA Mini Kit . RNase-R treatment was conducted by adding 2U RNase-R  per microgram of RNA, followed by a 1-h incubation at 37 °C. The RNA samples were reversely transcribed into cDNA by High-Capacity cDNA Reverse Transcription Kit . RPLPO gene was used as a reference gene, and the relative fold changes of indicated genes were calculated by 2The indicated cells were lysed by RIPA buffer and subsequently resolved on 10% SDS-PAGE. The membranes were probed by the following antibodies against β-catenin , Phospho-β-Catenin-Ser45 , Phospho-β-Catenin-Ser33/37/Thr41 , GSK3β , HA tag , and β-actin . The expression levels were visualized under enhanced chemiluminescence.The cDNA from Huh7 cells were amplified by PCR using a primer specifically targeting circβ-catenin. To confirm the head-to-tail splicing, Sanger sequencing was conducted by using sequencing primer 5′-GAAAAGCGGCTGTTAGTCA-3′.The SW620, Huh7, HepG2, and PLC/PRF/5 cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS. The parallel DNA sequences for shRNA were synthesized and cloned into pGLV31H1-GFP vector. The cancer cells  with stable knockdown of circβ-catenin were generated by using lentivirus vector pGLV31H1-GFP.http://sirna.wi.mit.edu/). The parallel DNA sequences were synthesized and cloned into pGLV31H1-GFP vector. The human circβ-catenin sequence was copied from human pcDNA3-β-catenin (Addgene #16828) and then inserted into pcDNA3.1 (+) circRNA mini vector (Addgene #60648) using the EcoRV and SacII sites. The sequence of the back-splicing junction has been modified when designing the primers, and thus, the back-splicing junction is not targeted by the shRNA. The overexpression effect was monitored by RT-PCR. The circβ-catenin expression vector with mutated ATG was generated by the Quick Change site-directed mutagenesis kit (Agilent Technologies). The primer sequences for plasmid construction were provided in the Additional file The short hairpin RNA (shRNA) targeting circβ-catenin was designed by online software .The cancer cells with stable knockdown of circβ-catenin and corresponding control cells were fixed with 70% ethanol at 4 °C overnight and then stained with staining buffer  at 4 °C for half hour. After PI staining, the cells were subject to flow cytometry analysis using LSR Fortessa and FACSDiva software .The liver cancer cells with stable knockdown of circβ-catenin and their corresponding control cells were grown to 100% confluence. Then, the pipette tips were utilized to scratch the monolayer cells. Twenty-four hours later, the open area was captured and calculated using ImageJ software .5 Huh7 and PLC/PRF/5 cells with stable knockdown of circβ-catenin, and their corresponding control cells were seeded into the upper chamber. Twenty-four hours later, the cancer cells on the upper compartment were removed. Then, the cells were stained with 0.2% crystal violet solution  for 15 min. Images were captured, and the cell number was counted by ImageJ .Transwell assay was conducted in a Boyden chamber with an 8.0-μm pore , and the upper surface of the chamber was coated with Matrigel. 1 × 10The cells were lysed by cold lysis buffer . The supernatant from cell lysates was incubated with indicated antibodies. Then, the protein G beads were incubated with the lysates. The beads were washed by cold lysis buffer, and the protein loading buffer was added to the beads, followed by western blotting analysis.2 fold change ≥ 1 were considered differentially expressed. The gene ontology analysis was carried out by online program DAVID (https://david.ncifcrf.gov/).The total RNA from PLC/PRF/5 cells with stable knockdown of circβ-catenin and control cells were isolated by Trizol  following the manufacturer’s protocol. The transcripts were sequenced using the BGISEQ-500 sequencing platform . After rRNA depletion, the RNA fragments were reversely transcribed into cDNA by using N6 random primer. The 5′ end of cDNA was phosphorylated, and 3′ end of cDNA was modified with stickiness “A.” Then, the cDNA was ligated with the adapter with sticky “T” at the 3′ end. Two specific primers are utilized to amplify the ligation DNA products. The DNA products were denatured by heat, and the single-stranded DNA was circularized by DNA ligase. Finally, the prepared DNA library was sequenced. Bowtie2 software was used to map clean reads to the reference gene, and HISAT software was utilized to map to the reference genome. Genes with a logMass spectrometry analysis was conducted by LTQ Velos Dual-Pressure Ion Trap Mass Spectrometer . An antibody which specifically recognized the N-terminus of β-catenin  was used to conduct a co-IP experiment to enrich the target proteins. After SDS-PAGE, the whole proteins were digested by using trypsin. Tandem mass spectrometry was used for protein sequencing following the manufacturer’s protocol. The identification of protein fragment was analyzed by the MASCOT database.+ membrane . Membranes were subsequently dried and crosslinked by ultraviolet. Pre-hybridization was performed at 62 °C for 2 h, and hybridization was performed at 62 °C for 16 h. The membrane was washed by SSC buffer with 0.1% SDS at 62 °C. After washing, the blots were visualized by the Typhoon scanner .Digoxin-labeled RNA probe targeting the exon region shared by circβ-catenin and β-catenin was prepared with DIG Northern Starter Kit . The target sequence of the RNA probe is 5′-GACACGCTATCATGCGTTCTCCTCAGATGGTGTCTGCTATTGTACGTACC-3′. Northern blotting assay was conducted by using Northern Max Kit from Ambion (Life Technologies) by following the manufacturer’s instructions. Electrophoresis of 15 μg total RNA was carried out on a 2% agarose gel and transferred to a Hybond-Nhttp://www.circbase.org/). The protein-coding capacity of circRNA was evaluated by using the online program http://202.195.183.4:8000/circrnadb/circRNADb.php. The predicted protein size was obtained from the website http://www.sciencegateway.org/tools/proteinmw.htm.The circular RNA sequence data of Wnt pathway-related genes was obtained from database circBase /2. For the in vivo lung metastasis experiments, 1 × 107 Huh7 cells were introduced into nude BALB/c mice by hydrodynamic tail vein injection. Two months later, the lung tissues were excised and subject to H&E staining to visualize the metastatic lesions within the lung. The lung metastatic lesions were counted under the microscope. For the orthotopic tumor model, the cancer cells were implanted into the liver of male C57BL/6 mice after anesthetization. Forty days later, the liver tissues were collected after the C57BL/6 mice were sacrificed and H&E staining was conducted to visualize the hepatic tumors. The animal handling procedures were approved by the institutional LASEC Guidelines in The Chinese University of Hong Kong. All animals were housed in an enriched environment and can freely access to food and water at 12:12-h light/dark cycle room.For the xenograft tumor model, 2 × 10HepG2 cells were transfected with indicated luciferase vectors. After lysed by lysis buffer, the luciferase activity was measured by the Dual-Luciferase® Reporter Assay System .Fifty paired HCC tissues and corresponding adjacent normal tissues were obtained from Prince of Wales Hospital  as previously reported , 5. Infot test. The correlation study was conducted using Pearson’s correlation. When the P value is less than 0.05, the results were considered to be statistically significant.The data are expressed as mean ± SD. The significance of difference was evaluated using Student’s Additional file 1:Table S1. A gene list of 143 Wnt pathway-related genes and their corresponding circular RNAs (XLSX 34 kb)Additional file 2:Figure S1–S8. with figure legends (PDF 668 kb)Additional file 3:Table S2. The RNA expression profile for deregulated genes after knockdown of circβ-catenin (XLSX 477 kb)Additional file 4:Table S3. The mass spectrometry analysis data (XLSX 78 kb)Additional file 5:Table S4. The primer and siRNA sequences (PDF 284 kb)


Cocos nucifera sheath (CS)/epoxy composites with graphene nano platelets (GNP). Laminates were fabricated through the hand lay-up method followed by hot pressing. GNP at different wt.%  were incorporated with epoxy resin through ultra-sonication. Kevlar/CS composites with different weight ratios  were fabricated while maintaining a fiber/matrix weight ratio at 45/55. Thermal degradation and viscoelastic properties were evaluated using thermogravimetric analysys (TGA), differential scanning calorimetric (DSC) analysis, and a dynamic mechanical analyser (DMA). The obtained results revealed that Kevlar/CS (25/75) hybrid composites at 0.75 wt.% of GNP exhibited similar thermal stability compared to Kevlar/epoxy (100/0) composites at 0 wt.% of GNP. It has been corroborated with DSC observation that GNP act as a thermal barrier. However, DMA results showed that the Kevlar/CS (50/50) hybrid composites at 0.75 wt.% of GNP exhibited almost equal viscoelastic properties compared to Kevlar/epoxy (100/0) composites at 0 wt.% GNP due to effective crosslinking, which improves the stress transfer rate. Hence, this research proved that Kevlar can be efficiently (50%) replaced with CS at an optimal GNP loading for structural applications.The aim of the present research work is to enhance the thermal and dynamic mechanical properties of Kevlar/ The ever-burgeoning need for humanity to produce lighter, tougher and cost-effective material has led to the development of composites . Ever siCocos nucifera sheath  could be a sustainable reinforcement in polymeric composites (epoxy) [Recent literature has made it evident that natural fibers can act as a promising and sustainable alternative to man-made synthetic fibers ,8. Recen (epoxy) . The ben (epoxy) . However (epoxy) ,12.Hybrid composites comprise more than two multi-scale fillers  embedded in a single or multiple polymeric matrix . The benThermogravimetric analysis (TGA) is extensively utilized to investigate the thermal degradation of polymeric composites, when the composite specimen is subjected to higher thermal loading . The theδ or damping factor is the ratio between E’ and E’’ [δ) while hybridizing coir and glass compared to coir/polyester composites. On the other hand, nano filler plays a vital role in the relaxation of macromolecular polymeric chains [Dynamic mechanical analysis (DMA) is the most widely used technique to measure the viscoelastic properties viz., storage modulus (E′), loss modulus (E″) and damping factor (Tanδ) while applying continuous sinusoidal loads ,29. Stor and E’’ . Several and E’’ . Devi et and E’’  studied  and E’’  also achc chains . Pedrazzc chains  found thFrom the literature analysis, it was observed that researchers devoted their efforts to replace synthetic fibers by natural fibers through hybridization. However, replacing synthetic fiber with natural fiber may affect the thermal and viscoelastic properties of hybrid composites. Hence, improving the thermal stability and viscoelastic properties of hybrid composites with an efficient nano filler is an interesting and effective research approach to produce high-performance composite structures at lower cost. The primary focus of this research work is to study the influence of incorporating different wt.% of GNP on the thermal degradation and dynamic mechanical properties of Kevlar/epoxy, Kevlar/CS/epoxy and CS/epoxy composites. In particular, previous research reported that Kevlar/CS laminates at 75/25 weight ratio in the absence of nano fillers exhibited almost equal thermal stability and viscoelastic properties compared to Kevlar/epoxy laminates . NonetheCocos nucifera sheath (CS) was procured from the villages of Serdang, Malaysia. The density of the CS varied between 1.37–1.50 g/cm3. The chemical compositions of CS were as follows: cellulose (21–22%), hemicellulose (42–43%), lignin (31–33%) and extractives (2–2.5%). The para aramid fabric used in this research was Kevlar 29. . D.E.R 331 epoxy resin with joint amine hardener (905-3S) were purchased from T.E.M. engineering bhd. . Graphene nanoplatelets (GNP) were procured from Sigma Aldrich, St. Louis, MO, USA. 2/g, bulk density: 0.2 g/cm3 and lateral dimension: 2–3 μm).A “Natural textile” and an “Agro wastes”, so called 3 was employed to fabricate the laminates. Initially, the mould was sprayed with a releasing agent (silicone spray), which improves the finishing and prevents the adhesion with the steel mould. The weight ratio of reinforcement and matrix was maintained to 45/55. On the other hand, the weight ratios of Kevlar/CS were maintained at 100/0 (S1), 75/25 (S2), 50/50 (S3), 25/75 (S4), and 0/100 (S5). Laminated composite samples were fabricated using the hand lay-up approach followed by hot pressing. A stainless steel mould with a dimensions of 150 × 150 × 3 mm2 environment (N2 flow rate: 50 mL/min). The analysis was carried out from room temperature to 900 °C (heating rate: 10 °C/min).The influence of adding GNP on the thermal stability of Kevlar/epoxy, CS/epoxy and hybrid Kevlar/CS composites was investigated using a thermo gravimetric analyzer  as per ASTM E1131-03 standards. As per the standard, the powdered composites (20 mg) were placed in an alumina crucible and underwent pyrolysis process in N2 flow rate: 50 mL/min) at a heating rate of 10 °C/min.Differential scanning calorimetry (DSC) testing was carried out using a DSC analyzer . Powdered composite sample of 20 mg was placed inside the crucible pan . Another crucible pan was employed as a reference without any sample. The DSC testing has been carried out from room temperature to 900 °C under a nitrogen purge .A dynamic mechanical analyser  was employed to evaluate the effect of adding GNP on the viscoelastic properties of different composites laminates. Dynamic mechanical analysis has been conducted in accordance with ASTMD 4065 standards. The dimensions of the test samples were 60 × 12.5 × 3 mmTGA analysis has been carried out for Kevlar/epoxy, CS/epoxy and Kevlar/CS hybrid composites at different GNP wt.%. It has been noticed that the addition of GNP enhanced the thermal stability of all the composite samples. It is well known that Kevlar/epoxy composites possess higher thermal stability while CS/epoxy composites degraded at lower temperature. Thermal stability of Kevlar/epoxy  and CS/epoxy composites  were also examined at different wt.% of GNP to compare the results with hybrid composites. The addition of GNP has shown only slight improvement in thermal stability for Kevlar/epoxy  and CS/epoxy composites . Fan et al.  found thNonetheless, hybridizing Kevlar and CS in the GNP modified epoxy composites will combine the properties of different constituents and results in higher thermal stability. Previous research has proven that the thermal stability of Kevlar/CS hybrid composites (75/25) is significantly closer to Kevlar/epoxy composites . HoweverCocos nucifera sheath (75%) at 0.75 wt.% of GNP (S4G3). Moreover, From g), curing temperature and decomposition temperature [g and decomposition temperature would be investigated through endothermic transition. The upper peak in the DSC curve represents the endothermic process while the lower peak indicates the exothermic process.perature ,45. The perature ,47,48. Ng of the epoxy [g to higher temperature (70–80 °C) at 0.25 and 0.5 wt.% of GNP. However, at higher GNP wt.% (0.75) there was not much improvement in Tg due to the agglomeration at the interface of the polymer and reinforcement which hinders the polymer cross linking [From the analysis the initial endothermic peak around 60–70 °C represents the The epoxy . Additio linking ,50. No e linking . The nex linking . The fin linking . The tem linking  when usig because of the polymeric chain movement. Movement in polymeric chain affects the stiffness as well as the fiber/matrix adhesion [g regions, while in the rubbery region overlapping E’ plots has proved that there was no notable change in E’ due to GNP [adhesion . During adhesion . From the to GNP .However, at 0.75 wt.% of GNP  there was not much improvement of storage modulus observed for S1, S2 and S3 laminates. From Cocos nucifera sheath content at an optimal GNP wt.% without affecting performance. From that point of view, S3G3 laminates (contains 50 wt.% CS at 0.75 wt.% of GNP) exhibited almost similar E’ value compared to S1G0. Hence, S3G3 laminates can efficiently replace the Kevlar/epoxy composites (S1G0).It is well known that man-made synthetic fiber-based composites possess higher storage modulus compared to natural fiber-based composites . Howeverg temperature range for all the laminates irrespective of the individual constituents. Moreover, all the laminates manifested two relaxations localized at Tg (65 °C to 75 °C) and 80–90 °C. Nevertheless, at higher temperature, overlapping of curves indicates that inclusion of GNP has no significant effect on the viscous dissipation [Loss modulus is a measure of released heat energy per cycle in a sinusoidal loading condition for a viscoelastic material . Figure sipation .g) could be obtained from the peak of both loss modulus as well as Tanδ plots. Nevertheless, Tg values observed in the loss modulus plot were more reliable compared to Tg from the damping plot [g obtained from E” and Tanδ are shown in g indicates the lack of interfacial adhesion [ing plot . Tg obtaadhesion .g could withstand higher load. This clearly indicates the improved load carrying capability of the composites after adding GNP. This is mainly attributed to the restriction in intermolecular movement of the polymeric chain [Addition of GNP at 0.25 wt.%  and 0.5 wt.%  drastically declined the Tanδ due to enhanced interfacial interactions a–c. Idicic chain ,27.However, at higher GNP loading  minimal deviation was observed compared to the composites with 0.5 wt.% of GNP . The Cole–Cole plot is a viable tool to interpret the relation between stored and dissipated heat energy for a viscoelastic material . MoreoveFrom The effects of adding GNP on the thermal stability and viscoelastic properties of Kevlar/epoxy, CS/epoxy and hybrid composites were evaluated as a function of temperature. The TGA results obtained confirmed that thermal degradation of S4G3 (hybrid composites) is almost closer to S1G0 (Kevlar/epoxy) due to GNP, which act as a thermal barrier and delayed the volatilization. From the dynamic mechanical analysis, it was observed that the storage modulus and loss modulus of S3G3 laminates (contains 50/50 weight ratio of Kevlar/ CS at 0.75 wt.% of GNP) exhibited almost similar E’ and E” value compared to S1G0 (Kevlar/epoxy). Also, S3G3 laminates exhibited higher compatibility between the multi-scale filler and epoxy with lower Tanδ. Moreover, S3G3 laminates exhibited rigid polymeric structure due to the restriction in intermolecular movement which enhanced the stress transfer rate and load-carrying capability. Overall, from the thermogravimetic and dynamic mechanical analysis it has been concluded that the hybrid composites S4G3 and S3G3 could efficiently replace Kevlar/epoxy (S1G0) polymeric structure at elevated temperature.


This research deals with the influence of different curing and post-curing temperatures on the mechanical and thermomechanical properties as well as the gel time of an epoxy resin prepared by the reaction of diglycidyl ether of bisphenol A (DGEBA) with an amine hardener and a reactive diluent derived from plants at 31 wt %. The highest performance was obtained for the resins cured at moderate-to-high temperatures, that is, 80  Since their early introduction in the 1930s, epoxy resins have been the most used thermosetting polymers available. Epoxy resins show a great range of inherent properties, resulting from their highly reactive epoxy groups located in the terminal chains . The resg) of the resultant epoxy resins [A potential advantage of epoxy systems is their capability to tailor their final properties on the basis of the selection of the processing conditions and parameters. In this context, choosing the type of cross-linking agent is one of the most effective methods. Different amine hardeners, anhydride hardeners, and acid hardeners have been tested to develop epoxy-type thermosetting polymers ,12,13,14y resins ,19. In ty resins ,20. Anoty resins ,22. Besiy resins .In recent years, a wide range of several renewable building blocks and green composites  have beeg, since some reactive groups, either from the epoxy resins or hardeners, do not react completely [g value [The improvement of the final properties of a given epoxy system can also be obtained by optimizing the curing process . In partmpletely . Thereaf[g value . Post-cu[g value ,33. This[g value ,35,36.The objective of this work is to study and optimize the temperature applied during curing and post-curing of a partially bio-based epoxy resin. The newly produced thermoset was evaluated in terms of its mechanical, morphological, rheological, and thermomechanical properties.® 1070 ECO and Resoltech® 1074 ECO, respectively, by Castro Composites . The epoxy resin is particularly made of a mixture of petroleum-derived DGEBA and a plant-based reactive diluent from vegetable oil epoxidation. As indicated by the supplier, the bio-based content of the cured resin is 31 wt %, as determined by ASTM D6866-12. The chemical structure of the main reactive components of the epoxy resin and the cross-linking/hardening system are shown in The commercial partially bio-based epoxy system was composed of an epoxy resin and an amine-based type hardener supplied as Resoltechwt/wt) in a glass vessel, following the manufacturer recommendations. To this end, the bio-based epoxy resin and the curing agent were thoroughly mixed manually for 5 min and then casted into a silicon mold. The resultant mixture has a density of 1.22 g·cm−3 and viscosity of 700 mPa·s and also, according to the manufacturer, its Tg can reach values of up to 73 The bio-based epoxy resin and the hardener were added under the stoichiometric ratio 100:35  with a 5 kN load cell. The cross-head speed was set at 10 mm·min−1. The impact strength was determined by the Charpy test, following the recommendations of ISO 179, in a Charpy pendulum from Metrotec  using a 1-J pendulum. Hardness was obtained following ISO 868 in a durometer 676-D from J. Bot Instruments . Tests were carried out at room temperature using, at least, six specimens of each sample.Flexural tests were performed according to ISO 178 on rectangular pieces with dimensions of 4  × 10 ×  80 mmThe morphology of the fracture surfaces of the bio-based epoxy resins obtained from the impact tests was analyzed by using field-emission scanning electron microscopy (FESEM) in a ZEISS ULTRA 55 FESEM microscope from Oxford Instruments . An acceleration voltage of 2 kV was used. All samples’ surfaces were previously coated with an ultrathin gold–palladium layer in a high-vacuum sputter coater EM MED20 from Leica Microsystem .3, were subjected to a heating program from 30 −1. The curing behavior was studied by means of a system of parallel plates of 25 mm diameter made for liquid samples, with a constant gap of 0.2 mm. These samples were extended over the preheated plates and subjected to an isothermal heating program at 70 G′) and the loss modulus (G″), in G′ = G″ conditions, according to Equation (1):C is a constant, and k is the reaction rate constant that can be parameterized through the Arrhenius expression, shown in Equation (2):A is the frequency factor, aE is the apparent activation energy, R refers to the universal gas constant, and T is the absolute temperature. By substituting Equation (2) in Equation (1), the following Equation (3) was obtained:The thermomechanical properties of the bio-based epoxy resins were evaluated by dynamic mechanical thermal analysis (DMTA) in an oscillatory rheometer AR-G2 from TA Instruments . Tests were carried out in torsion–shear conditions, with a special clamp system made for solid samples. The samples, sizing 4 × 10 × 40 mmThen, Equation (4) resulted by taking natural logarithms on both sides of Equation (3):aE was calculated through the slope of the linear fitting.Finally, by plotting fE) of the bio-based epoxy resin cured at 70 fE increased to values of 1260 ± 192 MPa and 2403 ± 210 MPa when the samples were cured at 80 fE and The flexural properties of the partially bio-based epoxy resins after curing and post-curing are gathered in  reduced . In relaa et al.  reportedfE and −2, after being cured at 70 −2, 13.3 ± 1.7 kJ·m−2, and 15.9 ± 3.7 kJ·m−2, respectively. The maximum energy absorption was also observed for the resin cured at 80 −2. Although fracture resistance tends to decrease when the cross-linking density increases [g. This effect is explained by the fact that the conformation configuration of the individual bonds is modified and, thus, the resultant free volume varies due to the rotation of the benzene rings in the epoxy resin.The hardness at the Shore D scale and the impact-absorbed energy values, obtained by the Charpy test, are also gathered in ncreases , this prncreases  reportedThe FESEM images of the surface fractures from the impact tests are shown in n et al.  indicate−3, for a curing temperature of 70 −3, for 80 −3, which can be ascribed to the formation of the aforementioned excessive cross-linked structure with a higher free volume. In this regard, Gupta and Brahatheeswaran [The densities of the partially bio-based epoxy resins after the different curing and post-curing processes were also evaluated in order to ascertain the effect of the thermal treatments on the resin free volume. The values of density are summarized in aterials . Howevereeswaran  proposedeeswaran . MoreoveG′ with time, shown in W of the bio-based epoxy resin increased and, thus, the values of G′ increased, improving the material elastic behavior. The curing processes of the partially bio-based epoxy resin carried out at 70 G′ and G″. G′max) values were achieved when the samples were completely cured. These values are also gathered in The gel time is one of the most important parameters from a manufacturing point of view since it delimits the change from the liquid to the solid state and it is noticed by an increase in the mixture viscosity. The gel point of these polymer networks occurred in a conversion around 0.7. This parameter can be estimated either when aE was obtained through the slope of the linear fitting of the plot of −1, having an excellent correlation coefficient  with temperature is shown in tan δ peak relates to the Tg of the biopolymer. The resins cured at 70 g values of approximately 62.5 ± 1.25 g [g values. For instance, the partially bio-based epoxy resins that were subjected to curing at 70 g values when compared with their counterpart resin without post-curing. In particular, the latter samples reached values of 90.5 ± 1.71 g was even more significant. In particular, the values obtained after post-curing at 150 g stabilization relates to a restriction of the mobility degree of the polymer chains. Therefore, the Tg values attained correlate with the previous density measurements, confirming the reduction of the sample free volume due to the successful extension of the cross-linking process by the formation of a more homogeneous structure. In addition, the partially bio-based epoxy resins subjected to post-curing at 125 g values than those post-cured at 150 The evolution of the damping factor (se of Tg . The effThe effect of the curing and post-curing conditions on epoxy resins prepared by the reaction of DGEBA with amine hardener and a bio-based reactive diluent at 31 wt % was analyzed by flexural tests, FESEM, and oscillatory rheometry at different isothermal heating rates. It was observed that the partially bio-based thermosetting resins cured and post-cured at higher temperatures tended to present higher mechanical and thermomechanical performance due to the formation of a highly cross-linked structure of lower free volume. However, interestingly, the samples cured at moderate temperatures and then post-cured at high temperatures unexpectedly developed improved toughness performance. In particular, it was observed that curing at 80


High aggressiveness is a hallmark of glioblastoma and predicts poor prognosis of patients with glioblastoma. The expression level of sortilin has been preliminarily reported to be elevated in high-grade glioma; however, the potential significance of sortilin in glioblastoma progression has not been elucidated. In this study, we investigated the oncogenic effect of sortilin in glioblastoma. Increased levels of sortilin were noted in the mesenchymal subtype of glioblastoma and highly aggressive subtypes of glioblastoma tissues and cell lines. In addition, high levels of sortilin predicted poor prognoses in patients with glioblastoma. Sortilin knockdown or inhibition with AF38469  significantly suppressed migration and invasion by inhibiting EMT-like mesenchymal transition in glioblastoma cells. Furthermore, we proved that sortilin promoted cell invasion mainly via Glycogen synthase kinase 3 beta (GSK-3β)/β-catenin/Twist-induced EMT-like mesenchymal transition in glioblastoma. Taken together, our results demonstrate a critical role of sortilin in glioblastoma invasion and EMT-like mesenchymal transition, indicating that sortilin contributes to glioblastoma progression. These data also highlight the dramatic antitumor effects of AF38469 in glioblastoma, suggesting that AF38469 is a potentially powerful antitumor agent for sortilin-overexpressing human glioblastoma. Although current multimodal therapeutic strategies for human GBM  have improved patient survival, the prognosis of patients with GBM is still dismal4. High aggressiveness is a hallmark of human GBM, which makes it difficult to be completely eradicated, resulting in relapse and death in patients with GBM. Although tumor invasion is a hot topic in the field, the mechanisms underlying GBM invasion are still not entirely understood. Therefore, elucidation of the molecular mechanisms underlying GBM invasion and development of novel and effective strategies for GBM treatment are urgently needed.Human glioblastoma (GBM) is the most common and aggressive form of malignant primary tumor in the central nervous system (CNS)5. Importantly, a recent report has confirmed that the mesenchymal subtype is closely related to the high invasive capacity of GBM6. In addition, WNT/β-catenin contributes to mesenchymal transition; WNT and β-catenin are expressed at high levels and are correlated with a significantly short survival time in patients with GBM8. In general, WNT/β-catenin is activated in GBM and contributes to tumor invasion by triggering the expression of EMT activators such as Twist, Snail, and ZEB19. Furthermore, accumulating evidence indicates that Twist, a downstream activator of WNT/β-catenin, is highly expressed in GBM and promotes cell invasion by regulating the expression of mesenchymal target genes11.Epithelial–mesenchymal transition (EMT) has been reported to induce epithelial cells to undergo numerous biochemical changes to switch to a mesenchymal phenotype, defined by an enhanced invasive capacity12. Sortilin is a member of the Vps10p sorting receptor family, which has important roles in various biological processes, such as transporting intracellular proteins, acting as a co-receptor for the 75 kDa neurotrophin receptor (p75NTR) or receptor tyrosine kinases (RTKs), and acting as a regulator of atherosclerosis14. Elevated expression of sortilin has been found in high-grade glioma and is positively correlated with the malignancy of glioma, suggesting that sortilin might have an important role in the progression of human glioma15. However, the potential significance of sortilin in GBM has not been elucidated.Our previous work has demonstrated that the overexpression of neurotensin (NTS) is closely linked with human glioma progression. The biological effects of NTS are triggered by its interaction with three distinct receptors NTSR1, NTSR2, and sortilin16. We found that AF38469 inhibited GBM invasion mainly through Glycogen synthase kinase 3 beta (GSK-3β)/β-catenin/Twist-induced mesenchymal transition in vitro and in vivo. Our results suggest that sortilin contributes to GBM progression and maybe a novel prognostic factor for GBM. Foremost, AF38469 could be developed as a promising therapeutic agent for human GBM.In this study, we investigated the expression levels of sortilin in the mesenchymal, classical, proneural, and neural subtypes of GBM. Bioinformatics analysis predicted that the expression level of sortilin was elevated in the mesenchymal subtype and a negative correlation was found between sortilin levels and the prognosis of patients with GBM. We used AF38469  to block the effects of sortilin on cell motility and mesenchymal transition in GBM2.Five human GBM tissues (including intratumor (IT) region, peritumor (PT) region, and normal brain (NB) region) were obtained from patients who are undergoing surgery at the Department of Neurosurgery, Daping Hospital of Army Medical University. Informed consent was obtained from all patients and the study was approved by the Ethics Committee of the Daping Hospital of Army Medical University. These GBM tissues were graded by at least two experienced clinical pathologists according to the World Health Organization classification system. GBM cell lines Rat (C6), Human , Mouse (GL261), and Human embryonic kidney cell line (HEK293T) were purchased from American Type Culture Collection. Short tandem repeat (STR) confirmation was conducted by the Institute Research of Surgery of Army Medical University. All cell lines were cultured in Dulbecco’s modified Eagle’s medium/F12 containing 10% fetal bovine serum and incubated at 37 ℃ in a humidified incubator with 5% COAF38469 was purchased from MedChem Express. SB216763, dimethyl sulfoxide (DMSO), and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma Aldrich. Lipofectamine 2000 was obtained from Invitrogen. Fluorescein isothiocyanate (FITC)-phalloidin was purchased from Life Technological. Rabbit anti-sortilin monoclonal antibody was purchased from GeneTex. Anti-GSK-3β, p-GSK-3β, β-catenin, T-cadherin, N-cadherin, vimentin, MMP-2, MMP-9, Twist, and Actin antibodies were purchased from CST.p-values were downloaded from the RGAVP.Kaplan–Meier analysis was conducted with the Gravedeel dataset and the Tumor GBM-TCGA-540 dataset from R2: Genomics Analysis and Visualization Platform (RGAVP), which includes 273 glioma cases and 485 cases of de novo GBM, respectively. The expression level of sortilin was investigated with the Gravedeel dataset from RGAVP, which includes 273 glioma cases with different histological grades. The expression levels of sortilin in four subtypes of human GBM were analyzed with the Tumor GBM-TCGA-540 dataset. The resulting BoxPlot, survival curves, and We purchased LV-Ctr and LV-Twist from Genechem, The GBM cells were infected with lentivirus particles or control lentivirus particles. Puromycin was used to induce stable cell line for subsequent assays. Small interfering RNA (siRNA) targeting sortilin, GSK-3β, and twist were purchased from RibiBio . The GBM cells were transfected with siRNA and cultured for 48 h before performing the assays according to the manufacturer’s instructions.4 cells in 100 μL of serum-free medium were seeded into the upper chamber. The lower chamber was filled with 600 μL of medium containing 5% fetal bovine serum. After 24 h of incubation, cells were removed from the upper surface. Invaded cells on the lower surface were fixed, permeated, and stained with 5% crystal violet (Beyotime). Invaded cells were counted in five randomly selected fields to quantify the invasive rate.Cells were seeded in six-well plates at 90% confluence and starved for 12 h. A wounding line was scratched with 10 μL pipet tip and the serum-free medium was added to the plates. Cells were incubated for 24 h. The migrated cells were monitored with Olympus inverted microscope and counted in five randomly selected fields to quantify the cell migration area. Transwell filters  were pre-coated with Matrigel (Corning). The cells were starved and 5 × 10+-K+-ATPase, and Actin were diluted to 1:1000. We calculated the three independent results by SPSS 13.0 and built graph by GraphPad 5.0.Cells were washed in phosphate-buffered saline and lysed in ice-cold lysis buffer for the whole-cell protein. The equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membrane. The membrane was blocked and incubated with primary antibodies. Specific antibodies against sortilin (1:3000), against GSK-3β, p-GSK-3β, β-catenin, T-cadherin, N-cadherin, vimentin, and Actin (1:1000), against MMP-2 and MMP-9 (1:5000), and against Twist (1:3000) were used to detect the corresponding protein. The membrane was washed and incubated with secondary antibodies. Immunoblots were detected by Western-Bright Sirius HRP Substrate Kit (Advansta) and exposed to ChemiDoc TMXRS + Image System (Bio-Rad). The Image J was used to analyze the relative expression level of protein. To detect the change of β-catenin expression in different subcellular compartments, we extracted membranal protein, cytoplasmic protein, and nuclear protein from whole-cell lysate by Nuclear and Cytoplasm Protein Extraction Kit (Beyotime) and Mem-PERTMPlus Membrane Protein Extraction Kit (ThermoFisher), then preformed western blotting assays to detect β-catenin and analyzed the results by Image J software. Antibody of β-catenin, PCNA, Na−ΔΔCt). Primers for PCR are as follow: SORT1:F:5′-GAAGTCGTGGAGGAAGAATCTTT-3′, R:5′-TGGTGTTGTCTGATCCCCATT-3′; GSK-3β:F:5′-GGCAGCATGAAAGTTAGCAGA-3′, R:5′-GGCGACCAGTTCTCCTGAATC-3′; β-catenin:F:5′-AAAGCGGCTGTTAGTCACTGG-3′, R:5′-CGAGTCATTGCATACTGTCCAT-3′; Twist:F:5′-GTCCGCAGTCTTACGAGGAG-3′, R:5′-GCTTGAGGGTCTGAATCTTGCT-3′; GAPDH:F:5′-ATTGACCTCAACTACATGGTTTACATG-3′, R:5′-TTGGAGGGATCTCGCTCCTGGAAG-3′.Total RNA extracted from the cultured cells by TRIzol reagent (Invitrogen) cDNA was reverse transcribed from 1.0 μg RNA with Prime Script RT Reagent Kit (TAKALA). The mRNA levels of sortilin, GSK-3β, β-catenin, twist, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) detected by Real-time PCR was performed with SYBR Premix Ex TaqTM Kit (TAKALA). The result was detected at CFX 96 Real-Time PCR Detection System (Bio-Rad). Fold changes in relative gene expression were calculated by comparative Ct method  and fluorescence-labeled secondary antibodies. The nucleus was stained with DAPI and examined with Zeiss LSM 780 Meta confocal microscope. The images were processed by ZEN software.6 U87 cells. The mice in the AF38469 group (n = 6) were intraperitoneally injected with AF38469 diluted in DMSO (0.01 g/day/kg) and the mice in control group (n = 6) were injected with same value of DMSO. The orthotopic xenografts model in nude mice was established by injection of 1 × 107 U87 cells into the right striatum of mice brains (n = 12). Magnetic resonance imaging (MRI) (Broker Biospec 7.0 Tesla Imaging System) was performed to detect tumor dimensions. After the first MRI detection to confirm successful transplantation, 12 mice was divided into two group, then intraperitoneally injected with AF38469 and same value of DMSO. All animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals, and all experimental protocols were approved by the Institutional Animal Care and Use Committee of Army Medical University.Immunodeficient athymic nude mice were purchased from the Experimental Animal Center of Army Medical University. Ectopic subcutaneous GBM transplantation model in nude mice was established by injection of 1 × 10t-test or one-way analysis of variance. Survival was analyzed by Kaplan–Meier method and compared by log-rank test by GraphPad Prism 5; *p < 0.05 was considered statistically significant.Statistical analyses were carried out using SPSS 13.0 statistical software. Statistical significance was calculated by Student’s n = 485), which includes 485 cases of de novo GBM. The Kaplan–Meier analysis showed that the 2-year survival rates of sortilin-low (n = 266) and sortilin-high (n = 219) GBM patients were 26% and 17%, respectively, and the 5-year survival rates were 9% and 1%, respectively . The result confirmed that high Soritlin expression indicated a significantly poor prognosis. In detail, Kaplan–Meier analysis showed that the 2-year survival rates for patients with high expression (58 cases) and low expression (215 cases) of Sortilin mRNA were 27% and 76%, respectively, and the 5-year survival rates for these patients were 13% and 51%, respectively Fig. . Meanwhiely Fig. . All graype Fig. .Fig. 1ThNext, we performed western blotting assays to detect the levels of sortilin in GBM cell lines and observed a high level of sortilin in U87 and A172 cells, which are highly aggressive Fig. . FurtherThe above data preliminarily indicate that sortilin is associated with the high aggressiveness of GBM. To investigate the biological effects of AF38469 on GBM cell motility, we performed wound-healing assays. Compared with DMSO, 400 nM AF38469 impaired the migratory capabilities of U87 and A172 cell lines Fig. . Next, wEMT-like mesenchymal phenotype is closely associated with the aggressive capacity of GBM and our data indicate that sortilin promotes the invasive ability of GBM. Thus, we speculated that sortilin is involved in the EMT-like transition of GBM. To confirm our hypothesis, we first detected the levels of EMT-related markers such as N-cadherin, vimentin, T-cadherin, and MMP-9 in the U87 cell line. Based on the results of western blotting, the levels of N-cadherin, vimentin, and MMP-9 in the AF38469 groups were markedly lower than those in the control groups; nevertheless, T-cadherin, a glial marker in the CNS, was strongly upregulated after treating with AF38469 Fig. . We also19. To further confirm these results, we also observed U87 and A172 cells changed their elongated and stretched, fibroblastic morphology toward minimal astrocytic-like morphology after sortilin was blocked by si-Sortilin. The amounts of filopodia sharply decreased after blocking sortilin by AF38469 or si-sor . U87 and A172 cells treated with AF38469 were more round and displayed decreased filopodia formation, whereas cells treated with DMSO had spindle-shaped morphology, displayed increase filopodia formation, and were more disorganized than the AF38469-treated cells Fig. . Based osor Fig. . TherefoWNT/β-catenin and Twist have been reported to be critical for EMT-like transition in GBM. To investigate whether sortilin enhances the EMT-like mesenchymal transition of GBM via WNT/β-catenin and Twist, we performed western blotting assays and found that AF38469 decreased the levels of p-GSK-3β, β-catenin, and Twist in U87 and A172 cells. We found similar results when Si-Sor blocked sortilin in both U87 and A172 cells Fig. . Further18. Our data confirm that AF38469 negatively modulates GSK-3β/β-catenin/Twist in GBM. To investigate whether GSK-3β/β-catenin/Twist is required for AF38469-inhibited GBM invasion and EMT-like transition, we first treated U87 cells with SB216763 and found this inhibitor significantly decreased T-cadherin expression, while increasing the levels of N-cadherin, vimentin, MMP-9, and Twist, indicating that SB216763 accelerated the EMT-like transition of GBM  or Si-GSK, suggesting that GSK-3β/β-catenin is positively regulated by sortilin in GBM. Moreover, WNT/β-catenin has been confirmed to be involved in GBM aggressiveness by triggering the expression of EMT activators, such as Twist, ZEB, Snail, and Slug. Indeed, our results demonstrated that AF38469 or Si-Sor decreased Twist expression and this effect could be reversed by SB216763 or Si-GSK, suggesting that sortilin positively regulates Twist expression via GSK-3β/β-catenin in GBM. Further results showed that the inhibitory effect of sortilin knockdown on GBM invasion and mesenchymal transition could be reversed by aGSK-3β inhibitor, indicating that GSK-3β/β-catenin is critical for sortilin-induced GBM invasion and mesenchymal transition. In addition, overexpression of Twist not only promoted GBM mesenchymal transition but also reversed the inhibitory effect of AF38469 on this process, implying that sortilin enhances GBM invasion and mesenchymal transition via Twist. The above data suggested that sortilin enhances GBM invasion mainly via GSK-3β/β-catenin/Twist-induced mesenchymal transition.20. Accumulating evidence indicates that sortilin is highly expressed in many cancers and is involved in the process of cancer development22. Some reports demonstrated a strong staining of sortilin in the cytoplasm and only 5–10% of sortilin on the cell surface24. Our study further confirmed this observation in GBM. These data show that sortilin might have diverse biological functions in cells. As an NTS receptor, sortilin is involved in the NTS-induced proliferation and migration of human microglial cells26. In addition, sortilin acts as a co-receptor with RTks and p75NTR to regulate numerous biological processes. Although sortilin is highly expressed in high-grade glioma and is positively correlated with glioma malignancy15, the biological function of sortilin in human GBM has not been clarified. In our study, we first demonstrated that sortilin promoted GBM invasion by enhancing mesenchymal transition both in vitro and in vivo. Compared with a series of sortilin inhibitors, such as AF40431, AF38469 has very high solubility and membrane permeability. These features make AF38469 a valuable molecular tool for investigating the biology of sortilin27. In this study, we observed that AF38469 efficiently repressed GBM invasion via GSK-3β/β-catenin/Twist-induced mesenchymal transition in vitro and in vivo.The biological effects of NTS are triggered by its interaction with NTSR1, NTSR2, and sortilin. Our previous study demonstrated that NTS and NTSR1 are overexpressed in human glioma, and that the activation of NTS/NTSR1 signaling is associated with the progression of glioma28. As one of RTK, EGFR strongly correlates with phosphatidyl inositol 3-kinase (PI3K)/AKT/GSK-3β29. Thus, sortilin may promote GSK-3β/β-catenin/Twist through repression EGFR/PI3K/AKT signaling in GBM, which will be investigated in our future study. In addition, although AF38469 has strong anti-GBM activity in vitro and in vivo, the ability of AF38469 to cross the blood–brain barrier (BBB) has not been investigated. However, according to previous research, AF38469 has low clearance and high solubility in DMSO, and high membrane permeability, which implies that AF38469 may as well be permeable to BBB16. In addition, it is critical for application of AF38469 in intracranial diseases such as human GBM, brain injury, and Alzheimer’s disease30. Moreover, we cannot ignore the potential toxicity of sortilin suppression observed in hepatocytes, adipocytes, and neurons33, and therefore an assessment of the potential risks and benefits of AF38469 is necessary.In this study, we focused on the GSK-3β/β-catenin/Twist pathway, because it is widely accepted as the most important signaling pathway in the mesenchymal transition of GBM. However, one limitation of our study is that the mechanism of sortilin-induced GSK-3β phosphorylation in GBM was not investigated in this study. Recently, study implies sortilin is a key regulator of epidermal growth factor receptor (EGFR) internalization and limits the EGFR signaling and loss of sortilin in tumor cells promoted cell proliferation by sustaining EGFR signaling at the cell surface, ultimately accelerating tumor growthWe revealed the oncogenic effects of sortilin in GBM aggressiveness and indicated that sortilin might be a potential prognostic factor for human GBM. We found that sortilin promoted GBM invasion via GSK-3β/β-catenin/Twist-induced mesenchymal transition, and that AF38469 could suppress GBM invasion and mesenchymal transition in vitro and in vivo Fig. . ImportaSupplementary figure legendsFigure S1Figure S2Cell line certification of A172Cell line certification of U87