Non-ampullary duodenal adenoma with activation of Wnt/β-catenin signalling is common in familial adenomatous polyposis (FAP) patients, whereas sporadic non-ampullary adenoma is uncommon. The adenoma-carcinoma sequence similar to colon cancer is associated with duodenal tumors in FAP, but not always in sporadic tumors. We obtained 37 non-ampullary duodenal tumors, including 25 adenomas and 12 adenocarcinomas, were obtained from biopsies and endoscopic resections. We performed immunohistochemistry for β-catenin, the hallmark of Wnt activation, and aldehyde dehydrogenase 1 (ALDH1), a putative cancer stem cell marker. In non-ampullary lesions, abnormal nuclear localization of β-catenin was observed in 21 (84.0%) of 25 adenomas and 4 (33.3%) of 12 adenocarcinomas. In the proximal duodenum, nuclear β-catenin was less frequent in both adenomas and adenocarcinomas. Gastric duodenal metaplasia (GDM) was observed only in the proximal duodenum. All adenomas with GDM were the gastric foveolar and pyloric gland types, and showed only membranous β-catenin. The intestinal-type adenomas had nuclear β-catenin in the proximal and distal duodenum. ALDH1-positive cells were more frequent in adenocarcinomas than adenomas. Nuclear β-catenin accumulation frequently occurred in ALDH1-positive cells in adenoma, but not in adenocarcinoma. In the non-ampullary proximal duodenum, Wnt/β-catenin pathway activation was more closely associated with adenomas than adenocarcinomas, and while it might cooperate with ALDH1 in adenoma, it does not in adenocarcinoma. The pathogenesis thus may differ between sporadic adenoma and adenocarcinoma of non-ampullary duodenal lesions, especially in the proximal and distal duodenum. APC) gene. Approximately 60% of duodenal adenomas develop in patients with FAP, while the remaining 40% are sporadic [Most small intestine adenomas, including the ampulla of Vater, develop in the duodenum. Among these, sporadic non-ampullary (except the ampulla of Vater) duodenal adenomas are uncommon , 2 compasporadic . Non-ampAPC gene, is a multifunctional cytoplasmic protein with a molecular weight of 92 kDa [APC and CTNNB1 gene mutation, is associated with early events in colon carcinogenesis in the model of the adenoma-carcinoma sequence [The Wnt/β-catenin pathway promotes organ development and tissue homeostasis , and disf 92 kDa . Wnt patsequence .+-dependent enzymes that metabolize endogenous and exogenous aliphatic and aromatic aldehyde molecules by oxidation to their corresponding carboxylic acids [ALDH genes [The aldehyde dehydrogenase (ALDH) superfamily constitutes a group of NAD(P)ic acids . The humDH genes . ALDH1 iDH genes , 16. ALDDH genes , 18.In the majority of colorectal cancers, the Wnt/β-catenin pathway is necessary for the self-renewal of cancer stem cells (CSCs) , 20. On Helicobacter pylori infection, celiac disease, or Crohn's disease [Helicobacter pylori infection [H. pylori infection compared with that in Western countries [Gastric duodenal metaplasia (GDM) is a histologically detectable mucosal change thought to evolve following  disease –27. The nfection . GDM hasnfection . Non-ampountries . Furtherountries .In this study, we evaluated β-catenin and ALDH1 expression in non-ampullary duodenal adenomas and adenocarcinomas using immunohistochemistry (IHC), and investigated the clinicopathological characteristics and non-ampullary duodenal carcinogenesis to provide a foundation for therapy development.P < 0.05). Additionally, no patients had a family history of Lynch syndrome.Thirty-seven patients were included in this study Table , 29 of wTo investigate the expression of β-catenin in non-ampullary duodenal adenoma and adenocarcinoma, we performed IHC with a β-catenin antibody. Normal epithelium in the duodenum showed strong membranous staining for β-catenin with a few nuclear β-catenin-positive cell in the bottom, and aberrant expression was observed in both adenoma and adenocarcinoma Figure . AbnormaNon-ampullary duodenal adenomas with high-grade dysplasia and large size (≥20 mm in diameter) indicate a high risk of progression to adenocarcinoma . In our Helicobacter pylori infection in most cases [We focused on the positional differences and histological subtypes of adenomas and adenocarcinomas of non-ampullary duodenal lesions. In the initial manifestation of GDM of our cohort, small patches of gastric foveolar cells appear near the tip of a villus Figure . The patst cases .To clarify what types of adenomas are associated with GDM, we performed IHC for CDX-2 , MUC5AC (a marker of gastric foveolar mucin), and MUC6 (a marker of gastric pyloric gland mucin). All 4 cases with GDM were gastric type adenomas. Two foveolar-type adenomas showed CDX-2(-), MUC5AC(+), MUC6(-), and 2 pyloric gland adenomas showed CDX-2(-), MUC5AC(-), MUC6(+) Figure . GDM is All 4 of our cases showed only membranous β-catenin expression. The other 21 adenomasshowed nuclear β-catenin expression and these were all intestinal-type adenomas identified with expressions of CDX2(+), MUC5AC(-), and MUC6(-)  part from the bulb to the periampulla and the lower  part from the periampulla to the descending duodenum. Nuclear β-catenin was frequent in adenoma and adenocarcinoma of the lower part and adenoma of the upper part Figure . HoweverWe investigated ALDH1 expression in non-ampullary duodenal adenoma and adenocarcinoma by IHC analysis. ALDH-positive cells were not detected in normal duodenal epithelium; however, ALDH1-positive cells were observed in adenomas and adenocarcinomas Figure . ALDH1 pTo investigate whether Wnt/β-catenin and ALDH1 activation occur in the same tumor cells of adenoma and adenocarcinoma, we performed both IF and IHC staining for distinct conclusion. In the IF assay, the co-localization of nuclear β-catenin and ALDH1 strong expression was observed in tumor cells of adenoma and adenocarcinoma Figure . In the In this study, we clarified the expression of β-catenin and ALDH1 in sporadic adenomas and adenocarcinomas in non-ampullary duodenal lesions by IHC. We have suggested the pathogenesis in sporadic non-ampullary duodenal adenoma and adenocarcinomaFigure . We demoWe did not include any cases of ampullary duodenal adenomas or adenocarcinomas because ampullary neoplasms may have combined features of neoplasms of the small intestine including duodenum, distal bile duct, and/or pancreas. Adenocarcinomas are particularly rare in non-ampullary lesions compared with adenomas.Nuclear β-catenin accumulation, a key effector of the Wnt signalling pathway, was frequent in adenoma rather than in adenocarcinoma. There were more ALDH1-positive cells with CSC potential in adenocarcinomas compared to adenomas. Further, nuclear β-catenin in these cells was more frequently co-expressed in adenomas rather than in adenocarcinomas. Our data suggest that Wnt/β-catenin activation might be associated with ALDH1-expressing CSCs in a subset of non-ampullary duodenal adenomas, i.e. intestinal type adenomas. There are similar findings in breast cancer, where ALDH1-positive cells show higher levels of Wnt/β-catenin signalling compared with ALDH1-negative cells . By contOur study showed nuclear β-catenin expression in 84% of adenomas and 33.3% of adenocarcinomas in non-ampullary duodenal lesions. A previous study observed aberrant β-catenin expression and nuclear/cytoplasmic translocation in 38.1% of duodenal adenocarcinomas, including both non-ampulla and ampulla of Vater . In contde novo type, i.e. gastric type, that occurs independent of the Wnt/β-catenin pathway, 2) adenoma-carcinoma sequence type, i.e. intestinal type, that develops dependent of the Wnt/β-catenin pathway like colon carcinogenesis. A possible difference between the non-ampullary duodenum and colorectum can be observed in the developing gut. Developmentally, the duodenum appears to share characteristics with the stomach, particularly the antrum, rather than the colorectum [H. pylori.In contrast to the typical adenoma-carcinoma sequence in the majority of colorectal carcinogenesis, the development of adenocarcinoma in non-ampullary duodenal lesions seems to have two types; 1) lorectum . In our Regarding colorectal cancers, the CSC population detected using ALDH1 enzymatic activity is typically very small, including 0.01–1% of the population of cancer cells . In contIn conclusion, nuclear β-catenin accumulation is more correlated with adenomas rather than adenocarcinomas in the non-ampullary proximal duodenum. The co-localization of nuclear β-catenin and ALDH1 is common in adenoma, and ALDH1 is highly expressed in adenocarcinoma rather than in adenoma. The pathogenesis may differ in sporadic adenoma and adenocarcinoma of the non-ampullary duodenum, especially the proximal and distal duodenum.Tumor specimens were obtained from patients through biopsy, EMR, or ESD at Gifu University Hospital between 2008 and 2015, and Gifu Municipal Hospital between 2012 and 2015. Informed consent was obtained from all patients.Using hematoxylin and eosin-stained sections, adenoma was diagnosed when the tumor was circumscribed and found to be composed of tubular structures showing intraepithelial neoplasia. Adenocarcinoma was diagnosed when the tumor invaded into the lamina propria or through the muscularis mucosae. On biopsy, samples contained areas suggestive of true invasion. Adenocarcinoma without invasion was diagnosed when increased architectural distortion with glandular crowing and prominent cellular atypia and pleomorphic, hyperchromatic, pseudostratified nuclei were observed. The diagnoses were confirmed by at least two pathologists.All paraffin-embedded tissues were cut into 4 μm-thick serial sections and deparaffinized. These sections were stained with hematoxylin and eosin or used for IHC. For IHC, the sections were placed in citrate buffer (pH 6.0), and then autoclaved at a chamber temperature of 121°C for 1 min to retrieve the antigen. The sections were then rinsed and blocked in 3% hydrogen peroxide in methanol for 10 min to remove endogenous peroxidase. Non-specific binding sites were blocked in 0.01 M phosphate-buffered saline (pH 7.4) containing 2% bovine serum albumin  for 40 min. They were incubated with β-catenin antibody  and ALDH1A1 antibody  overnight at 4°C.Primary antibodies were detected using a biotinylated anti- mouse and rabbit IgG  for 30 min at room temperature, followed by incubation with avidin-coupled peroxidase  for 30 min. The bound complex was visualized using diaminobenzidine liquid chromogen , and counterstained with hematoxylin. As a negative control, the primary antibody was replaced with normal serum IgG at a similar dilution.For double-staining IHC, β-catenin and ALDH1 were used as primary antibodies. Signal amplification was performed using alkaline phosphatase-biotin complex (Vectastain), followed by a chromogenic reaction with an alkaline phosphatase substrate kit  and diaminobenzidine.For immunofluorescent (IF) staining, after treatment with primary antibodies, samples were incubated for 30 min at room temperature with FITC- or rhodamine-conjugated secondary antibodies . DNA was labeled with 4′, 6-diamidino-2-phenylindole. Histological evaluation was performed by two experienced pathologists (H.T. and A. N.), who were blinded to the clinical data.Staining of β-catenin was graded as either nuclear (moderate or strong staining of the nucleus) or membrane (very weak or negative in the nucleus). ALDH1 staining intensity scores were rated on a scale of 0–4 according to the percentage of positive tumour cells .In double staining, percentages of nuclear β-catenin- and/or ALDH1-positive cells in adenoma and adenocarcinoma were obtained by manually counting positively stained cells in 10 representative fields of adenomatous or cancerous regions, in which nuclear β-catenin translocation was most frequently observed under 400× high-power magnification.P < 0.05 was considered to indicate statistical significance.Statistical analysis and graphical presentation were performed using the GraphPad Prism 6.0 package . The Mann-Whitney U, Fisher's exact test, or chi-square test was used to test the relationship between categorical variables as appropriate.


PP2A is composed of a scaffolding subunit (A), a catalytic subunit (C) and a regulatory subunit (B) that is classified into four families including B, B′, B′′ and B′′′/striatin. Here, we found that a distinct PP2A complex regulates NF‐κB signalling by dephosphorylation of IKKβ, IκBα and RelA/p65. The PP2A core enzyme AC dimer and the holoenzyme AB′′′C trimer dephosphorylate IKKβ, IκBα and RelA, whereas the ABC trimer dephosphorylates IκBα but not IKKβ and RelA in cells. In contrast, AB′C and AB′′C trimers have little effect on dephosphorylation of these signalling proteins. These results suggest that different forms of PP2A regulate NF‐κB pathway signalling through multiple steps each in a different manner, thereby finely tuning NF‐κB‐ and IKKβ‐mediated cellular responses. IκB, inhibitor of kappa BIKKβ, inhibitor of kappa B kinase βIKKβKN, kinase‐negative IKKβ mutantNF‐κB, nuclear factor kappa BPPPs, protein phosphatasesNuclear factor kappa B (NF‐κB) is a critical transcription factor that regulates many cellular and organismal processes including immune and inflammatory responses, cellular growth and cell survival. The NF‐κB signalling pathway is regulated by the phosphorylation of several proteins including inhibitor of kappa B (IκB) kinase β (IKKβ), an inhibitory protein IκBα and an essential subunit of NF‐κB RelA/p65 Aberrant activation of NF‐κB is linked to various diseases such as inflammatory disorders and cancer. Thus, there are numerous regulatory mechanisms at multiple levels to ensure the tight control of NF‐κB activity Among these phosphatases, PP2A is the most abundant, constituting approximately 1% of total cellular proteins A previous RNAi screen revealed that PP2A plays an important role in the regulation of NF‐κB signalling and identified the core enzyme subunits, Aα, Aβ, Cα and Cβ, as negative regulators of the NF‐κB signalling pathway in mouse astrocytes m L‐glutamine at 37 °C.cDNA‐encoding Aα, Aβ, Cα, Cβ, Bα, Bβ, Bγ, Bδ, B′α, B′β, B′γ, B′δ, B′ε, B′′β, Strn and Strn3 subunits of PP2A were amplified from a human cDNA library by PCR. The cDNAs were inserted into pRK‐HA and pRK‐Flag expression vectors. Expression plasmids encoding IKKβ, kinase‐negative IKKβ mutant (IKKβKN) and IκBα have been reported previously −1 TNF‐α for 18 h. After lysing cells with buffer from the assay system, luciferase activity was analysed following the manufacturer's instructions.Nuclear factor kappa B activity was estimated by Dual‐Luciferase Reporter Assay System (Promega) using pNF‐κB and pRL‐TK Luciferase reporter plasmids. After transfection of 0.01 μg NF‐κB reporter plasmids with or without 0.01 μg expression plasmids of various PP2A subunits, IKKβ, IκBα and/or RelA into GP2‐293 cells in collagen‐coated 96‐well dishes, cells were incubated in the presence or absence of 50 ng·mLm Tris/Cl, pH 7.5, 150 mm NaCl, 1 mm EGTA, 10 mm MgCl2, 60 mm β‐glycerophosphate, 1 mm Na3VO4, 1 mm 4‐amidino phenyl methyl sulfonyl fluoride, 50 KIU·mL−1 aprotinin, 20 μg·mL−1 pepstatin, 20 μg·mL−1 leupeptin, 2 mm DTT and 1% Triton X‐100. After centrifugation at 16 000 × g for 20 min at 4 °C, the supernatants were used as cell lysates. The cell lysates were subjected to SDS/PAGE and then gel‐separated proteins were transferred to PVDF membranes (Millipore) and subjected to immunoblotting using the SuperSignal West Pico Chemiluminescence System (Pierce). Antibodies used were as follows: anti‐Flag (M2) , anti‐HA , anti‐IκBα , anti‐RelA/p65 , anti‐phospho‐IκBα , anti‐phospho‐RelA/p65(S468) , anti‐phospho‐RelA/p65(S536) , anti‐PP2A , anti‐PP2A , anti‐IKKβ  and anti‐phospho‐IKKα/β .GP2‐293 cells in collagen‐coated 12‐well dishes were transfected with 0.2 μg expression plasmids of various PP2A subunits, IKKβ, IκBα and/or RelA. After 24‐h transfection, cells were washed with PBS and solubilized with buffer A consisting of 20 mvia anti‐Flag (M2) agarose beads (Sigma). IKKβ proteins were eluted in a buffer containing 20 mm Tris/Cl, pH 7.5, 150 mm NaCl, 2 mm DTT, 1% Triton X‐100 and 3 × Flag peptides (Sigma) from anti‐Flag‐agarose beads with ultra‐free Centrifugal Filter Units (Millipore). For the dephosphorylation assay of IκBα and RelA, cells were transfected with plasmids encoding Flag‐RelA, HA‐IκBαRR and IKKβEE, and then phosphorylated IκBαRR and RelA protein complexes were purified from cell lysates via anti‐Flag (M2) agarose beads. PP2A proteins were purified from GP2‐293 cells following transfection with plasmids encoding the Flag‐tagged Aα subunit and HA‐tagged Cα subunit, together with or without HA‐tagged B subunits including Bα, Bβ, Bγ, Bδ, B′α, B′β, B′γ, B′δ, B′ε, B′′β, Strn and Strn3. PP2A core enzyme composed of Aα and Cα, and PP2A holoenzyme composed of Aα, Cα and various B subunits were purified from cell lysates via anti‐Flag (M2) agarose beads with ultra‐free Centrifugal Filter Units in the previously described buffer. Phosphorylated IKKβ proteins (0.001 μg) or protein complexes (0.001 μg) of phosphorylated IκBαRR and RelA were incubated with purified PP2A (0.001 μg) for 30 min at 37 °C, and then reaction mixtures were subjected to SDS/PAGE. Gel‐separated proteins were transferred to PVDF membranes and subjected to immunoblotting using anti‐phospho‐IKKα/β, anti‐phospho‐IκBα and anti‐phospho‐RelA antibodies, respectively.For the dephosphorylation assay of IKKβ, GP2‐293 cells in 10‐cm dishes were transfected with expression plasmids encoding Flag‐IKKβ, and then phosphorylated IKKβ was recovered from cell lysates PP2A is composed of three subunits scaffolding (A), regulatory (B) and catalytic (C) subunit Fig. A. Cells To investigate the involvement of the PP2A in regulation of NF‐κB signalling, we constructed expression plasmids encoding each subunit of the B, B′′, B′′ and B′′′/Strn family (Fig. We next investigated the effects of distinct B subunits on phosphorylation of the IKKβ activation loop. Cells were transfected with expression plasmids encoding IKKβ and plasmids encoding Aα, Cα and each B subunit, and phosphorylation of the activation loop was investigated by immunoblotting. Expression of the AC core enzyme markedly suppressed IKKβ phosphorylation in a phosphatase activity‐dependent manner Fig. A. The ACTo reveal the effects of each B subunit on the NF‐κB/IκBα complex, we transfected cells with expression plasmids encoding IκBαRR, RelA and IKKβEE, together with plasmids encoding Aα, Cα and each B subunit. Then, the effects of PP2A on IKKβEE‐mediated phosphorylation of IκBαRR and RelA were investigated by immunoblotting. Expression of the AC core enzyme suppressed phosphorylation of IκBαRR and RelA in a phosphatase activity‐dependent manner Fig. C. AC, ABThen, we investigated the effects of distinct B subunits on phosphorylation of endogenous NF‐κB signalling proteins in TNF‐α‐stimulated cells. Cells were transfected with expression plasmids of PP2A, and then stimulated with TNF‐α. AC and AB′′′C suppressed TNF‐α‐induced phosphorylation of IKKβ and RelA, whereas ABC, AB′C and AB′′C had little effect Fig. E. AC, ABin vitro. Expression plasmids encoding Flag‐tagged Aα, HA‐tagged Cα and HA‐tagged B subunits were transfected in cells, and PP2A complexes of various isoforms of B subunit family were purified from cells by using Flag‐agarose beads (Fig. in vitro, AC and AB′′′C preferentially dephosphorylate IKKβ and RelA, and AC, ABC and AB′′′C preferentially dephosphorylate IκBα in vivo, suggesting that intracellular mechanisms may relate to the preference of AC, ABC and AB′′′C to dephosphorylate IKKβ, IκBα and RelA.Dephosphorylation of IKKβ, IκBαRR and RelA was analysed by using purified PP2A enzyme ads Fig. A. Phosphads Fig. B. Followads Fig. C. Then, ads Fig. D. Followads Fig. E. These in vitro and in vitro, we investigated intracellular mechanisms of the preference of AC, ABC, AB′′C and AB′′′C to dephosphorylate NF‐κB signalling proteins. The effects of B subunits on the interaction between PP2A and IKKβ were analysed by immunoprecipitation assay. Cells were transfected with plasmids encoding Flag‐tagged CαD85N, HA‐tagged Aα, HA‐tagged B subunits and HA‐tagged IKKβ. PP2A complexes were immunoprecipitated by using Flag‐agarose beads from cells, and the association of IKKβ proteins to the PP2A complexes was analysed by immunoblotting (Fig. To resolve the discrepancies in the dephosphorylation of IKKβ and RelA between in vitro and in vivoin vivoMany studies have revealed important and complicated roles of PP2A in IKKβ regulation. For example, it has been reported that PP2A suppresses IKKβ activity NF‐κB signalling is not uniformly regulated by PP2A but rather is subjected to specific regulation by distinct PP2A complexes at multiple steps. In contrast to AC and AB′′′C, which dephosphorylate IKKβ, IκBα and RelA, ABC dephosphorylates IκBα but not IKKβ and RelA in cells. These results indicate that ABC suppresses NF‐κB activity without inhibition of IKKβ. A recent study revealed that IKKβ not only activates NF‐κB through the phosphorylation of IκBα but also regulates many cellular functions by phosphorylating various proteins in an NF‐κB‐independent manner via A subunits of PP2A in the complexes. STRIPAK complexes have a critical role in protein dephosphorylation and act as important regulators of multiple vital signalling pathways, including the Hippo pathway, mitogen‐activated protein kinases and cytoskeleton remodelling. Recent studies suggest that the dysregulation of STRIPAK complexes correlates with human diseases including cancer Among the four classes of B subunit families, the B′′′/Strn family proteins only facilitate dephosphorylation of three proteins including IKKβ, IκBα and RelA. Members of the B′′′/Strn family are evolutionarily conserved and have critical roles in biological processes such as development and cell growth PP2A is a confirmed tumour suppressor protein that is genetically altered or functionally inactivated in many cancers HK and YT designed and performed the experiments, interpreted the study, and wrote the paper. KO, MK, YN, ET and TH performed the experiments.


Great progress has been achieved in the study of the aerobic glycolysis or the so-called Warburg effect in a variety of cancers; however, the regulation of the Warburg effect in Nasopharyngeal carcinoma (NPC) has not been completely defined.Gene expression pattern of NPC cells were used to test associations between Chibby and β-catenin expression. Chibby siRNAs and over-expression vector were transfected into NPC cells to down-regulate or up-regulate Chibby expression. Loss- and gain-of function assays were performed to investigate the role of Chibby in NPC cells. Western blot, cell proliferation, Glucose uptake, Lactate release, ATP level, and O2 consumption assays were used to determine the mechanism of Chibby regulation of underlying targets. Finally, immunohistochemistry assay of fresh NPC and nasopharyngeal normal tissue sample were used to detect the expression of Chibby, β-Catenin, and PDK1 by immunostaining.We observed that Chibby, a β-catenin-associated antagonist, is down-regulated in nasopharyngeal carcinoma cell lines and inhibits Wnt/β-Catenin signaling induced Warburg effect. Mechanism study revealed that Chibby regulates aerobic glycolysis in NPC cells through pyruvate dehydrogenase kinase 1(PDK1), an important enzyme involved in glucose metabolism. Moreover, Chibby suppresses aerobic glycolysis of NPC via Wnt/β-Catenin-Lin28/let7-PDK1 cascade. Chibby and PDK1 are critical for Wnt/β-Catenin signaling induced NPC cell proliferation both in vitro and in vivo. Finally, immunostaining assay of tissue samples provides an important clinical relevance among Chibby, Wnt/β-Catenin signaling and PDK1.Our study reveals an association between Chibby expression and cancer aerobic glycolysis, which highlights the importance of Wnt/β-catenin pathway in regulation of energy metabolism of NPC. These results indicate that Chibby and PDK1 are the potential target for NPC treatment.The online version of this article (10.1186/s13046-018-0769-4) contains supplementary material, which is available to authorized users. One of the most important hallmarks of cancer is aerobic glycolysis or the so-called Warburg effect. The Warburg effect was first described by Warburg over 90 years ago and states that cancer cells heavily rely on glycolysis for energy metabolism even under normal oxygen concentrations . ConsequThe canonical Wnt signaling pathway plays a central role in normal development and tumorigenesis , 7. The Chibby was identified as β-catenin antagonist in a protein-protein interaction screen using the bait of C-terminal region of β-catenin in 2003 . Chibby Clinical Chibby, β-Catenin, PDK1 protein levels were detected from primary human nasopharyngeal cancer or normal tissue. All samples were obtained from the First Affiliated Hospital of Xiamen University with patient consent and institutional review board approval. These samples were subsequently de-identified to protect patient confidentiality.6 cells were injected subcutaneously into the dorsal thighs of mice. Tumor growth was monitored regularly for 6 weeks, then the tumor volume was calculated every week. All mice were kept under specific pathogen-free conditions at Xiamen University Laboratory Animal Center  in accordance with institutional guidelines. This study was approved by the local Ethical Committee of Xiamen University.Four-week-old female BALB/c nude mice were used. A total of 4 × 10p). p < 0.05 was considered statistically significant.Date were analyzed using GraphPad Prism software. Data are presented as the means ± standard error. The Student’s t-test (two-tailed), Fisher’s exact test, and Pearson’s r were used to compare data and to calculate their probability value  cells . MoreoveTo examine whether Chibby inhibits Wnt/β-catenin signaling-induced PDK1 expression, we overexpressed β-catenin in CNE1 and HK-1 cells. As expected, PDK1 expression levels were significantly increased, whereas simultaneous overexpression of Chibby was found to almost completely compromise Wnt/β-catenin activity Fig. . We nextSince our results have clearly demonstrated that Chibby inhibits Wnt/β-catenin signaling-induced PDK1 expression and aerobic glycolysis in NPC cells, we further explored whether Chibby and PDK1 are critical for Wnt/β-catenin signaling-induced NPC cell proliferation. First, we knocked down PDK1 in β-catenin-overexpressed CNE1 cells. Cell growth analysis revealed that knockdown of PDK1 diminished the promoting effect of β-catenin on cell proliferation Fig. . Similarr = − 0.578, p < 0.001, Fig. r = − 0.642, p < 0.001, Fig. r = 0.649, p < 0.001, Fig. To investigate whether Chibby is associated with Wnt/β-catenin signaling and PDK1 in NPC patients, 45 fresh normal tissue samples and 45 NPC tissue samples were used to detect the expression of Chibby, β-catenin, and PDK1 by immunostaining. As indicated by the representative samples in Fig. Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors and is reported as an endemic disease with high prevalence in Southeast Asia, particularly in South China , 23. TheWnt/β-catenin signaling has been implicated in the mediation of cancer cell metabolism via multiple mechanisms . SpecifiGiven the strong evidence for regulation of PDK1 protein expression by Chibby through Wnt/β-catenin signaling, we asked whether Chibby, Wnt/β-catenin signaling and PDK1 were correlated in primary human NPC specimens. We used 45 pairs of fresh NPC samples with normal tissues to detect the expression of Chibby, nuclear β-catenin, and PDK1 by immunostaining. Indeed, we observed a strong inverse correlation between Chibby and nuclear β-catenin or PDK1 levels and a strong correlation between nuclear β-catenin and PDK1, which supports the results observed in in vitro experiments.Taken together, our findings identified Chibby as a negative regulator of proliferation that suppresses NPC aerobic glycolysis via the inhibition of Wnt/β-catenin signaling in vitro and in vivo. The modulation of this molecular process may be a method of inhibiting NPC cell growth by restoring Chibby expression to interfere with cell metabolism. In particular, the PDK1 may become a new target for further inhibitor design to interfere with Wnt/β-catenin dependent NPC progression.Additional file 1:Supplementary Methods. (DOCX 19 kb)


Dissolving microneedles (DMNs) are microscopic needles capable of delivering encapsulated compounds and releasing them into the skin in a minimally invasive manner. Most studies indicate that encapsulating therapeutics in DMNs is an efficacious approach; however, the importance of evaluating the activity of encapsulated compounds, during the fabrication process, has not been examined in detail. Conducting an analysis of thermal, chemical, and physical stress factors, including temperature, pH, and the interaction of the polymer and therapeutics mixture during preparation, is essential for retaining the activity of encapsulated therapeutics during and after fabrication. Here, we optimised the thermal, chemical, and physical parameters for the fabrication of exendin-4 (Ex-4)–encapsulated DMNs (Ex-4 DMNs). Ex-4, a peptide agonist of glucagon-like peptide (GLP) receptor, is used for glycaemic control in patients with type 2 diabetes. Our findings indicate that optimising the parameters involved in DMN fabrication retained the activity of Ex-4 by up to 98.3 ± 1.5%. Ex-4 DMNs reduced the blood-glucose level in diabetic mice with efficiency similar to that of a subcutaneous injection. We believe that this study paves way for the commercialisation of an efficient and minimally invasive treatment for patients with type 2 diabetes. This can lead to a series of severe syndromes such as cardiovascular disease, hypertension, stroke, blindness, and increased risk of cancer2. Exendin-4 (Ex-4), a 39-amino acid incretin hormone that acts as a glucagon-like peptide-1 receptor agonist (GLP-1 agonist), is used for glycaemic control in patients with type 2 diabetes3. Long-term treatment with Ex-4 reduces food intake, lowers gastric motility4, and suppresses glucagon secretion5, without causing severe side effects, in patients with type 2 diabetes6.Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus, disrupts the normal action and secretion of insulin in the body7. Daily injections create a large amount of needle waste, which may result in needle-stick injury, blood-borne virus transmission, and needle recycling costs10. To improve patient compliance and delivery efficacy of Ex-4, various studies have evaluated the encapsulation of Ex-4 in microspheres, long-acting conjugate polymers, and nanoparticles14. However, due to concerns about stability and toxicity, hypodermic injection remains the most common delivery route for patients with type 2 diabetes. Therefore, the development of a safe, painless, and efficient delivery system for Ex-4 is important for improving patient compliance and overcoming limitations associated with SC injection15.Ex-4 is generally administered by subcutaneous (SC) injection on a daily basis, which may result in pain, needle phobia, infection, and inconvenience to the patient17. DMNs provide an efficient and accurate delivery system for encapsulated therapeutics, and overcome the limitations of hypodermic injections18. Most studies suggest that DMNs can be used as a rapid and efficient transdermal delivery system for small-molecule drugs, nanoparticles, and biomolecules such as insulin, influenza vaccine, and the human growth hormone23. Despite this potential for using DMNs as highly efficient delivery vehicles for Ex-4, only a few reports have evaluated the encapsulation of Ex-4 in DMNs (Ex-4 DMNs)25. Furthermore, these studies have mainly focussed on the design and delivery of Ex-4 DMNs rather than addressing the activity of encapsulated Ex-4 during the fabrication process and storage period. Therefore, we hypothesised that optimising the parameters involved in DMN fabrication will help maintain the activity of Ex-4 at a high level. Optimising such a minimally invasive system will improve compliance of patients with type 2 diabetes and help commercialise Ex-4 DMNs as an efficient replacement for the currently used SC.Dissolving microneedles (DMNs), which consist of a biodegradable polymer and therapeutic, are used for delivering encapsulated compounds, after insertion into the skin, in a minimally invasive manner26. We analysed the thermal, chemical, and physical factors that can affect the activity of Ex-4 DMNs. Based on these data, we optimised the fabrication conditions for Ex-4 DMNs for fabrication of Ex-4 DMNs in which the activity of encapsulated Ex-4 can be maintained at up to 98.3 ± 1.5%. The in vivo delivery comparison of SC injection and Ex-4 DMNs suggested that DMNs possess efficiency similar to that of SC in reducing the levels of blood glucose. We believe that compared with the current delivery systems, Ex-4-DMNs provide efficient delivery and can improve patient compliance, thereby offering an effective alternative for treating patients with type 2 diabetes.In this study, we used the most recent technique for DMN fabrication, in which the activity of encapsulated compounds is highly conserved during the fabrication process27. Therefore, we evaluated the effects of thermal conditions on the activity of Ex-4 at 4, 25, 60, and 80 °C for up to 90 min   Fig. . Our finTo confirm this, and to find the optimal concentration of CMC at which the activity of Ex-4 is retained the most, we examined the effects of CMC concentration on the activity of Ex-4. As expected, our results indicate that the activity of Ex-4 was retained at all the tested concentrations of 4, 8, 10 and 12% CMC (w/v) Fig. . Overall34. We found that CMC concentrations of 4 and 8% were not suitable for the fabrication of DMNs, whereas both 10 (1.47 ± 0.15 Pa.s) and 12% (2.75 ± 0.09 Pa.s) possessed the required viscosity  were used as controls and were not detected for up to 15 min post-insertion (data not shown). These findings show that the DMNs, fabricated in this study, can efficiently deliver encapsulated Ex-4 through the skin barrier.We used the Franz diffusion cell, which mimics the natural blood circulation in the body, to assess the release profile of Ex-4 DMNs through the skinTo evaluate the activity of Ex-4 DMNs post-fabrication, we used ultra-performance liquid chromatography (UPLC) to compare the initial activity of Ex-4 in 10% CMC (before DMN fabrication) with that of extracts of Ex-4 DMN (after DMN fabrication) Fig. . The ret38. Therefore, we examined the secondary structure of Ex-4, before and after DMN fabrication, using circular dichroism (CD). The Ex-4 DMN group showed the same peak as that of Ex-4, with a negative spectra range of 210–247 nm. The area under the curve of 10% mixture of Ex-4 CMC, and that of Ex-4 DMN extract, were smaller than that of Ex-4  were highly instrumental in maintaining the activity of encapsulated Ex-4. We selected 10% CMC because its viscosity afforded the fracture force of 0.36 N and because it rapidly dissolved inside the skin 15 min post-application. With respect to storage, Ex-4 DMNs showed no significant reduction in activity after 8 weeks of storage at a low temperature. Our findings indicate that Ex-4 DMNs can potentially replace the currently used SC injections, because they can efficiently reduce blood-glucose levels in patients with type 2 diabetes and are convenient to use. In addition to introducing a novel method for delivering Ex-4, our results highlight the importance of evaluating factors that may affect the activity of encapsulated therapeutics during each step of the DMN fabrication process.184H282N50O60S) was purchased from the AnyGen Co. . Biodegradable polymer carboxymethylcellulose , phosphate buffered saline , HEPES buffer (pH 5.5), buffer solution , potassium bromide (KBr), formic acid solution, acetonitrile (ACN), 4, 5- dimethylthiazol-2-yl-2,5- diphenyl tetrazolium bromide (MTT), and Avertin  were purchased from Sigma-Aldrich . Human embryonic kidney 293 T (HEK293T) cells were purchased from ATCC . Male C57BLKS/J db/db mice  were purchased from Japan SLC, Inc. . The Ex-4 enzyme-linked immunoassay (ELISA) kit was purchased from Phoenix Pharmaceuticals, Inc. .Ex-4  equipped with an Eclipse Plus C18 RRHD column . Chromatographic separation was achieved using the mobile phases (A) 0.1% formic acid in deionised water and (B) 0.1% formic acid in acetonitrile, at the following gradient elution: 0.0–1.0 min, A 95%, B 5%; 1.0–3.0 min, A 95-35%, B 5–65%; 3.0–3.5 min, A 35%, B 65%; 3.5–3.8 min, A 35–95%, B 65-5%. The conditions for the analysis were set at a flow rate of 0.45 ml/min, injection volume of 10 μl, and column temperature at 25 °C with the detection wavelength of 276 nm.Ex-4 was dissolved in PBS (pH 7.4) at the concentration of 100 μg/ml and incubated in a 4, 25, 60, and 80 °C water bath for 0–90 min to assess the effects of temperature. To evaluate the effects of pH on the activity of Ex-4, the Ex-4 solution, at the concentration of 100 μg/ml, was stored for 6 h at 4 °C in buffers at pH 4, 5.5, 7.4, 8, 9, and 10. The relative activity of Ex-4 was measured, via UPLC, with a standard curve obtained using the concentration of 0–1 mg/ml Ex-4 with R squared = 0.99. Ex-4 dissolved in PBS (pH 7.4) buffer was set as 100% active.To prepare the CMC mixture, powdered CMC was dissolved in PBS at the concentrations of 4, 8, 10, and 12% (w/v). These solubilised mixtures were then homogenised, via centrifugation, for 10 min at 4 °C and 4500 rpm using an Allegra X-30R centrifuge . The pH of each CMC mixture was measured using an ST3100 pH meter . The viscosity of each CMC mixture was measured via a Viscometer  at the shear rate of 200/s, at 25 °C.−1 by accumulating 32 scans at a resolution of 4 cm−1. The bioactivity of Ex-4 was measured using an ELISA assay kit according to the manufacturer’s instructions. The mixture solutions of CMC and Ex-4 were diluted at the concentrations of 0.01 to 100 ng/ml, and the absorbance was calculated using a Victor X5 microplate reader  at 560 nm.To analyse the interaction of CMC and Ex-4, 10 mg/ml of Ex-4 was mixed with the 4, 8, 10, and 12% (w/v) CMC polymer, and examined by a Vertex70 FT-IR spectroscope . All the samples were measured using potassium bromide (KBr) pellets. Transmittance spectra were acquired in the range of 400–4000 cm26. Blank DMNs, without Ex-4, were also fabricated using the same method and imaged via LEICA M165 FC . The mechanical fracture force of a single DMN was measured by a force test machine . DMN was attached to the stainless-steel station, and the sensor probe was pressed against the DMN at a speed of 3.6 mm/min at 0.02 N via an axial force.Ex-4 DMN was fabricated in 3 × 3 arrays using 10 mg/ml of Ex-4 mixed with 10% CMC (w/v) via centrifugal lithography2 sections) and stored at −10 °C until analysis. Tissue samples were first embedded in OCT compound  at −30 °C, and then sectioned at 25 μm per slice. The sections were next stained with hematoxylin for 5 min, eosin for 1 min, dehydrated through a series of alcohol gradients and cleared by xylene .A single Ex-4 DMN was applied to hairless pig cadaver skin (1 mm thick 2.5 × 2.5 cm2 sections) for 5, 10, and 15 min. Images of DMNs were acquired using a LEICA M165 FC microscope. The profile of Ex-4 release from DMNs was examined using a Franz diffusion cell . The diffusion cell was filled with 7 ml PBS (pH 7.4); then, the Ex-4 DMN patches were inserted into the hairless pig cadaver skin. After securing with a pinch clamp, the contents of the chamber were thoroughly mixed via a magnetic stirrer at 500 rpm, and 2 μl samples were acquired from the receptor at 0, 20, 40, 60, 90, 120, and 150 min. The amount of Ex-4 was quantitated by a NanoVue UV/Visible spectrophotometer  at the absorbance of 208 nm. The value of the blank DMN was used as negative control.The Ex-4 DMN patches were applied to hairless pig cadaver skin (1 mm thick 2.5 × 2.5 cmThe activity of Ex-4 (at the concentration of 100 μg/ml), that of a mixture solution of Ex-4 and 10% CMC (w/v), and that of the extract of Ex-4 DMN was examined via UPLC and CD at different stages of the DMN fabrication process. CMC (10% w/v) was used as negative control. CD was conducted using a Jasco CD Spectrophotometer J-815  with a path length of 10 mm. The parameters were set at a scanning speed of 100 nm/min, band-width of 0.1 nm, wavelength range of 190–260 nm, and the baseline was corrected using a blank solvent.The cytotoxicity of Ex-4 DMNs was evaluated via a MTT cell proliferation assay. HEK293T cells were cultured for 24 h in 3 ml of Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% foetal bovine serum (FBS) and 1% antibiotics, at a density of 105 cells/well in 6-well plates, at 37 °C, in a humidified atmosphere of 5% CO2. After treating HEK293T cells with 200 μl solutions of blank DMN and Ex-4 DMN dissolved in 200 μl PBS, the cells were incubated for 24 h. Cells were then incubated with 300 μl of MTT reagent (1 mg/ml) for 1 h, then the medium was removed, and the cells were treated with 1 ml of dimethyl sulfoxide (DMSO) per well. Cell viability (%) was calculated at the absorbance of 560 nm using a Victor X5 microplate reader . The viability of the PBS-treated control cells was set as 100%.Each Ex-4 DMN was packed and fully sealed into a plastic bag, with no light exposure, and stored at 4 and 25 °C for 8 weeks. Then, the DMNs were dissolved in 1 ml PBS and activity was measured using UPLC.Male C57BLKS/J db/db mice (6–7 weeks old) were selected for the Ex-4 bioactivity study. First, the mice were anaesthetised with Avertin (125 mg/kg) and the fur on their backs was removed with a shaver. Before the experiment, the mice were fasted overnight, with free access to water, and randomly divided into the following groups: (a) blank DMN group (negative control); (b) SC injection group ; (c) Ex-4 DMN patch group (DMNs loaded with 10 μg Ex-4); (d) Ex-4 DMN stored for 8 weeks at 4 °C group . Blood-glucose levels were measured by collecting 20 μl of blood samples from the tail vein at 1, 2, 3, 5, 7, 9, 11, 13, and 15 h post-administration; measurements were conducted using AccuChek Go . Plasma was then separated by centrifugation at 13,000 rpm for 10 min. The serum samples were immediately frozen at − 80 °C until analysis. The Ex-4 concentrations in plasma samples were determined using an ELISA kit at the R squared value of 0.97. All animal studies were conducted in accordance with the guide for the care and use of laboratory animals, and in accordance with the procedures approved by the Committee of the Department of Laboratory Animal Medicine .All the experimental values are expressed as mean ± S.E. Statistical differences were considered to be significant at a P value < 0.05 (Student’s t-test).All relevant data are available within the manuscript and supplementary information.Supplementary information


CX3CR1-deficient mice however still develop allodynia, albeit delayed; thus, additional underlying mechanisms emerge as VCR accumulates. Whilst both patrolling and inflammatory monocytes express CX3CR1, only inflammatory monocytes express CCR2 receptors. We therefore assessed the role of CCR2 in monocytes in later stages of VCR-induced allodynia.A dose-limiting side effect of chemotherapeutic agents such as vincristine (VCR) is neuropathic pain, which is poorly managed at present. Chemokine-mediated immune cell/neuron communication in preclinical VCR-induced pain forms an intriguing basis for the development of analgesics. In a murine VCR model, CX2- or CX3CR1-deficient mice. In CX3CR1-deficient mice, the CCR2 antagonist, RS-102895, was also administered. Immunohistochemistry and Western blot analysis were employed to determine monocyte/macrophage infiltration into the sciatic nerve as well as neuronal activation in lumbar DRG, whilst flow cytometry was used to characterise monocytes in CX3CR1-deficient mice. In addition, THP-1 cells were used to assess CX3CR1-CCR2 receptor interactions in vitro, with Western blot analysis and ELISA being used to assess expression of CCR2 and proinflammatory cytokines.Mechanically evoked hypersensitivity was assessed in VCR-treated CCR2 signalling plays a mechanistic role in allodynia that develops in CX3CR1-deficient mice with increasing VCR exposure. Indeed, the CCR2 antagonist, RS-102895, proves ineffective in mice possessing functional CX3CR1 receptors but reduces VCR-induced allodynia in CX3CR1-deficient mice, in which CCR2+ monocytes are elevated by VCR. We suggest that a novel interaction between CX3CR1 and CCR2 receptors in monocytes accounts for the therapeutic effect of RS-102895 in CX3CR1-deficient mice. Indeed, we observe that CCR2, along with its ligand, CCL2, is elevated in the sciatic nerve in CX3CR1-deficient mice, whilst in THP-1 cells (human monocytes), downregulating CX3CR1 upregulates CCR2 expression via p38 MAP kinase signalling. We also show that the CX3CR1-CCR2 interaction in vitro regulates the release of pronociceptive cytokines TNF-α and IL1β.We show that CCR2/CCR2 signalling plays a crucial role in VCR-induced allodynia in CX3CR1-deficient mice, which arises as a result of an interaction between CX3CR1 and CCR2 in monocytes.Our data suggests that CCLThe online version of this article (10.1186/s12974-018-1116-6) contains supplementary material, which is available to authorized users. Chemotherapy-induced painful neuropathy (CIPN) is a dose-limiting side effect of chemotherapeutic drugs, which is poorly managed by analgesics at present . CurrentMost chemotherapeutic agents do not cross the blood-brain barrier but are able to penetrate the blood-nerve barrier, where they accumulate in dorsal root ganglia (DRG) and peripheral nerves, causing toxicity that is intensified by the absence of a lymphatic system in the endoneurial compartment –7. Treat3CR1-expressing monocytes into peripheral nerves, where they differentiate into macrophages and activate TRPA1 channels on sensory neurons via the release of reactive oxygen species [3CR1-mediated activation of macrophages specifically, as well as inhibition of p38 MAPK, a downstream target of CX3CR1 activation, also blocks the development of paclitaxel-induced allodynia [One chemotherapeutic agent associated with neuropathic pain is the vinca alkaloid, vincristine (VCR), used most commonly in the treatment of lymphomas and leukaemias . In a pr species . An incr species . Communi species , 12. Furllodynia .3CR1 receptors in monocytes/macrophages as a prophylaxis for VCR pain, and, indeed, pain associated with other chemotherapeutic agents, a critical caveat remains: CX3CR1-deficient mice still develop VCR-induced allodynia, albeit delayed [3CR1 in monocytes is dynamic and alternative mechanisms emerge with increased exposure to VCR. In this study, we attempt to elucidate such a mechanism that could account for the eventual onset of VCR-induced allodynia in CX3CR1-deficient mice.Despite the promise of targeting CX delayed . It therlow) and high levels of CX3CR1 (CX3CR1high), are predominantly associated with homeostatic functions [high) and relatively low levels of CX3CR1 (CX3CR1low). Unlike patrolling monocytes, inflammatory monocytes express the chemokine receptor CCR2 (CCR2+). In the sciatic nerve, CCR2 receptor is activated by CCL2, which is expressed by endothelial cells, monocytes/macrophages and sensory neurons [2/CCR2-regulated fashion, releasing a multitude of factors, many of which exert pronociceptive functions [2+ phenotype [Circulating monocytes can, very broadly speaking, exist as two phenotypes—‘patrolling’ or ‘inflammatory’, each of which expresses distinct markers . Patrollunctions . Inflamm neurons –17. Influnctions . It is whenotype , 20.2/CCR2 signalling in inflammatory monocytes/macrophages in VCR-induced allodynia—specifically at later stages of treatment, when the role of CX3CR1 is less pertinent. Given that the CCR2 receptor is also expressed in sensory neurons [2-mediated neuronal activation in order to differentiate between the roles of CCR2 signalling in sensory neurons and monocytes. As well as attempting to elucidate the role of CCR2+ monocytes/macrophages in VCR-induced allodynia in CX3CR1-deficient mice, we also investigate a potential interaction between CX3CR1 and CCR2 expression in immortalised human monocytes (THP-1 cells). Furthermore, we assess the regulation of pronociceptive cytokines from THP-1 cell downstream of the CX3CR1-CCR2 interaction, which could constitute a potential monocyte-derived signal that mediates monocyte communication with nociceptive neurons, which are known to express cytokine receptors [In this study, we therefore investigate the role of CCL neurons , we alsoeceptors .Experiments were performed in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 and local animal care and use guidelines. Male and female mice  were used. They did not differ with respect to thresholds. Mice were randomly assigned to groups. Each group contained approximately equal numbers of age-matched mice of both sexes.For Cx3cr1-gfp (green fluorescent protein) mice, an original breeding stock was a kind gift from Steffen Jung . Cx3cr1-gfp heterozygous mice were generated and genotyped as described . For CcrStatic mechanical withdrawal thresholds were assessed as described previously . Briefly2 receptor belonging to the spiro-piperidine class, was dissolved in dimethyl sulphoxide (DMSO) at 10 mg/ml and diluted in sterile saline; 20 mg/kg/day was injected i.p. for 5 days [Vincristine sulphate  was dissolved in sterile saline. 0.5 mg/kg/day VCR was injected intraperitoneally (i.p.) using a 25-g needle for two 5-day cycles (days 0–4 and 7–11) with a 2-day break as previously described ). RS-102r 5 days  during tMice, under pentobarbital anaesthesia, were transcardially perfused with saline, followed by 4% paraformaldehyde (PFA). The lumbar enlargement, bilateral L3–5 DRG, and sciatic nerves were excised. Tissue was post-fixed for 4 h and then dehydrated in 30% sucrose in PBS before being embedded in optimum cutting temperature-embedding medium (VWR) and frozen on dry ice. Fifteen-micrometer cryosections were mounted onto Superfrost Plus slides (VWR). Sections were permeabilised for 15 min at room temperature in 0.1% PBS-Triton X-100 (Sigma-Aldrich). Slides were blocked in 0.1% PBS-Triton X-100 + 3% BSA (Sigma-Aldrich) for 1 h at room temperature before being incubated in primary antibody at 4 °C for 16 h. Slides were washed thrice in 0.1% PBS-Triton X-100, before being incubated with the appropriate fluorescently tagged secondary antibody for 1.5 h at room temperature. Slides were washed again and then mounted using FluorSave™  and visualised under a Zeiss LSM710 confocal microscope (Zeiss). Five sections per mouse were randomly selected and analysed blind as described previously .2 in the sciatic nerve, anti-CCL2  and anti-rabbit Alexa 568  were used. P-ERK visualisation in DRG was carried out using anti-p-ERK  followed by anti-rabbit Alexa 568 (1 in 1000 Invitrogen). GFP did not require amplification. In all cases, control staining was performed in which no primary antibody was applied.To visualise microglia, anti-Iba1  and anti-rabbit Alexa Fluor 488  were used. For F4/80 expression, anti-F4/80  was used followed by anti-rat Alexa 488 or 568 . For CCL3VO4 ; and 10 mg/ml proteinase inhibitor (Roche)) before being incubated at 4 °C with agitation for 2 h. Samples were centrifuged at 4 °C and 13,000 rpm for 20 min. Supernatant was used for Western blot. For THP-1 cultures, medium was spun at 2000g for 5 min at 4 °C. The pellet was washed, re-suspended in radioimmunoprecipitation assay (RIPA) buffer, vortexed and kept on ice for 30 min, and then spun at 13,000 rpm at 4 °C for 10 min. The supernatant was collected. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay (Pierce). Twenty micrograms of protein per sample was separated on a 15% SDS-PAGE gel and transferred onto a nitrocellulose membrane which was performed according to the manufacturer’s instructions . Blots were blocked for 1 h at room temperature in 0.1% TBS-Tween-20 (TBST) + 5% skimmed milk before being incubated in primary antibody (F4/80), CCL2 (anti-mouse or human), CCR2 (anti-mouse or human)  and p-ERK  at 4 °C overnight along with a loading control . After washing in 0.1% TBST, blots were incubated for 1 h at room temperature with HRP-conjugated secondary antibody . Blots were washed before being developed with SuperSignal™ West Femto substrate (Thermo Fisher Scientific). Bands were visualised using BioSpectrum© Imaging and quantified using Quantity One .Sciatic nerves were fresh-dissected, snap-frozen and stored at − 80 °C. Tissue was homogenised in ice-cold RIPA buffer (20 mM tris(hydroxymethyl)aminomethane; 10 mM NaF; 150 mM NaCl; 1% nonyl-phenoxylpolyethoxylethanol; 1 mM phenylmethanesulfonyl fluoride; 1 mM Na2. Peritoneal cells were then elicited by injecting saline into the peritoneal cavity. Lavages were collected and centrifuged at 2400 rpm for 10 min. Pellets were suspended in red phenol-free DMEM (Gibco) supplemented with 10% heat-inactivated foetal bovine serum (HI-FBS), 1% pen/strep and 1% sodium pyruvate. Cells were incubated with conjugated antibodies (CD45.1-Pacific Blue (BioLegend); F4/80-PE (eBioscience); Alexa Fluor® 647 anti-mouse CD192 (CCR2) Antibody (BioLegend); Ly-6C APC (eBioscience)) on ice for 30 min and then washed twice with PBS containing 1.5% BSA before being re-suspended prior to analysis. Cells were gated and analysed with LSRFortessa™ (BD bioscience) and FlowJo software (Tree Start).Mice were sacrificed by a rising concentration of CO3CR1 siRNA (Insight Biotechnology) or control SignalSilence®siRNA (New England Biolabs) 24 h after seeding, and assays were performed 48 h post-transfection. Efficiency of CX3CR1 knockdown was determined using Western blot. In some experiments, cells were pre-treated, 1 h before transfection with siRNA, with SB203580 or PD98059 . For CCL2 stimulation, cells were treated for 3 h with human recombinant CCL2 (Cambridge Bioscience) at 10, 50 and 100 ng/ml in HEPES buffer.THP-1 cells were a kind gift from Mauro Perretti . Cells were cultured in RPMI 1640 with 10% FBS and 2 mM glutamine (Sigma-Aldrich) and maintained in upright T25 tissue culture flasks. For all experiments, cells were cultured in 24-well plates. Cells were transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific) with CX2 expression was measured using human chemokine C-C-motif receptor 2 (CCR2) ELISA Kit (2B Scientific) according to the manufacturer’s instructions. For cytokine expression, a multi-analyte ELISA for proinflammatory cytokines (Qiagen) was performed on culture medium to assess which cytokines were upregulated. ELISA was then performed specifically for TNFα and IL1β (Abcam) according to the manufacturer’s instructions.CCRt test. All data are shown as mean ± SEM, and data were considered significant when p < 0.05.All data were analysed using SPSS . Behavioural data were analysed by repeated measures (RM) two-way ANOVA, followed by Tukey’s test. All other in vivo data were analysed by one-way ANOVA, followed by Tukey’s test. All in vitro data was analysed using Student’s paired 2 receptor signalling plays a role in VCR-induced allodynia, CCR2-deficient mice (CCR2RFP/RFP) were treated with two 5-day cycles of VCR [2+/RFP), littermate controls . During the second cycle of VCR however, CCR2-deficient mice had significantly higher thresholds, thus reduced allodynia, relative to heterozygous controls  as opposed to prophylactic (VCR cycle 1) dosing was derived not only from the apparent effect of CCR2 deficiency on VCR-induced allodynia during the second VCR cycle specifically but also from previous observations that whilst the development of VCR-induced allodynia is orchestrated by CX3CR1 signalling in monocytes, other mechanisms appear to regulate allodynia during the second VCR cycle [3CR1 heterozygous mice were given RS-102895 during the second VCR cycle, there was still no change in VCR-induced allodynia  mice with two cycles of VCR in the presence of RS-102895 (or vehicle) during the second VCR cycle, when allodynia manifests in CX3CR1-deficient mice [3CR1-deficient mice displayed a delayed onset of allodynia in response to VCR treatment relative to heterozygous littermates. Whilst CX3CR1 heterozygous mice developed significant allodynia within 24 h of the first VCR dose , a marker for neuronal activation and downstream target of CCR2 signalling [3CR1-deficient and heterozygous mice .Given the effect of RS-102895 treatment on CXic cells , 28, we ncy Fig. , b. In aice Fig. , b. Immuges Fig. , d. In o2 antagonist (RS-102895) reduces VCR-induced allodynia and monocyte/macrophage infiltration in sciatic nerves in CX3CR1-deficient mice during cycle two specifically and (ii) VCR induces CCL2 elevation in monocytes/macrophages. However, whether VCR allodynia is associated with peripheral nerve infiltration of CCR2-expressing monocytes, which provide a cellular target for RS-102895, has yet to be determined. We therefore considered evaluating monocytes/macrophages in peripheral nerve tissue specifically; however, the yield obtained during isolation was suboptimal for reliable characterisation. We therefore used peritoneal lavages as a surrogate source of monocytes [2 receptor is indeed detectable in both control and lipopolysaccharide (LPS)-stimulated peritoneal cells using both Western blot and FACS analysis of Ly6C+ CCR2+ cells  treatment with a CCRt RS-10285 reduces2 receptor is detectable, we measured peritoneal CCR2+ monocytes (F4/80− cells) in CX3CR1-deficient and heterozygous mice following two cycles of VCR using FACS analysis. We observed that in both genotypes, two cycles of VCR treatment significantly increased F4/80−Ly6C+CCR2+ cells relative to saline-treated controls , was not significantly higher than those observed following CX3CR1 receptor downregulation alone  is a dose-limiting side effect that jeopardises the success of cancer therapy . The nee2 receptor signalling in monocytes infiltrating the sciatic nerve plays a role in VCR-induced allodynia in CX3CR1-deficient mice during the second cycle specifically. We begin to elucidate a novel interaction between CX3CR1 and CCR2 in monocytes and demonstrate in vitro that it regulates the release of proinflammatory cytokines that have been previously shown to modulate neuronal mechanisms underlying chronic pain [In this study, we elucidate a mechanism underlying pain associated with vincristine (VCR) treatment and present evidence to suggest that CCRnic pain , 29.2-deficient mice, VCR-induced allodynia and concurrent monocyte infiltration into the sciatic nerve develop to the same severity and within the same time frame as CCR2 heterozygous littermates but are both significantly reduced during the second VCR cycle. Our findings from CCR2-deficient mice however is not enough to confirm a role for CCR2 signalling in monocytes per se in VCR-induced pain. CCR2 mediates the egression of monocytes from the bone marrow into the bloodstream where they survey the environment surrounding tissue [2 signalling in macrophages in the sciatic nerve specifically. Indeed, pharmacological inhibition of CCR2 affords a very different scenario biologically to CCR2 deficiency and as such is accompanied by different behavioural and biological manifestations. Specifically, when we administered the CCR2 antagonist RS-102895 to VCR-treated mice during the second cycle, we observed that neither VCR-induced allodynia nor monocyte infiltration into the sciatic nerve was reduced.We initially show that in CCRg tissue , 31. It 3CR1-deficient mice. Indeed, in keeping with this, CCR2 receptor inhibition in CX3CR1-deficient mice specifically has also been found to be protective in other pathological conditions such as macular degeneration [Our pivotal observation however was that RS-102895 administration during the second VCR cycle did significantly reduce established VCR-induced allodynia and monocyte infiltration into the sciatic nerve of CXneration .2+ monocytes in the peritoneal cavity was exacerbated in CX3CR1-deficient mice and (ii) RS-102895 treatment significantly reduced the VCR-induced increase in CCR2+ monocytes in CX3CR1-deficient mice specifically. Taken together, these data are suggestive of an interaction between CX3CL1/CX3CR1 and CCL2/CCR2 chemokine/receptor systems in monocytes that could underlie the onset of VCR-induced allodynia in CX3CR1-deficient mice.In addition we found that the (i) VCR-induced increase in CCR3CR1 receptor and CCL2 have been reported previously in other contexts. Indeed, in addition to our observation that CCL2 is elevated in the sciatic nerve of CX3CR1-deficient mice, the expression of CCL2 has also been shown to be elevated in mononuclear phagocytes in the retina in CX3CR1-deficient mice [2 receptor expression is also altered downstream of CX3CR1 downregulation in vitro. Specifically, when we downregulated CX3CR1 expression in immortalised human monocyte THP-1 cells, we observed a significant upregulation in both CCL2 and CCR2. Pharmacological inhibition of p38 MAPK but not MEK prevented this effect, and thus, we suggest that downregulation of CX3CR1 results in upregulation of the CCR2 receptor via p38 MAPK signalling.Interactions between CXent mice . Here, w3CR1-mediated regulation or CCL2 and CCR2 expression in monocytes, the converse interaction has also been observed previously. Specifically, CCL2 stimulation of monocytes has been found to increase CX3CR1 receptor expression at monocyte membranes, most likely due to the stimulation of receptor translocation from intracellular stores as opposed to the stimulation of de novo expression [2-mediated activation of p38 MAPK, with pharmacological inhibition of p38 MAPK, attenuating the effect [3CR1 and CCR2 receptors in monocytes is reciprocal and that expression of CX3CR1 and CCR2 is mutually regulated.As well as CXpression . This ree effect . This da3CR1 deficiency being associated with an increase in CCL2-CCR2 expression, VCR treatment was also found to increase CCL2. Elevated CCL2/CCR2 signalling in CX3CR1-deificient mice under basal conditions could explain the efficacy of RS-102895. The basal elevation of CCL2/CCR2 signalling alone in CX3CR1-deficient mice is not likely to be sufficient to cause allodynia as withdrawal thresholds did not vary between saline-treated CX3CR1-deficient and heterozygous mice. However, the potentially additive effect of CX3CR1 deficiency and two cycles of VCR on CCL2/CCR2 expression could indeed result in susceptibility to hypersensitivity. This could account for the onset of allodynia in CX3CR1-deficient mice at later stages of VCR treatment and the therapeutic benefit of CCR2 inhibition, which could also be attributed to increases in CCR2+ monocytes found in CX3CR1-deficient mice. Indeed, CCL2/R2 signalling in sensory neurons is a well-established mediator of pain. The elevation of CCR2+ monocytes and CCL2 expression in sciatic nerves that we observe in CX3CR1-deficient mice, in addition to VCR-induced increases in CCL2, is likely to cumulatively increase activity of the CCL2/CCR2 signalling axis in sensory neurons, thus activating pain pathways [As well as CXpathways –34.3CR1 is mainly found in monocytes/macrophages, the CCR2 receptor is expressed by other cell types. Elucidating the role of CCR2 signalling in monocytes/macrophages specifically is therefore a more intricate process than unravelling the role of CX3CR1 signalling in monocytes. For instance, the CCR2 receptor is also expressed in sensory neurons [3CR1-deficient mice to inhibition of CCR2 in monocytes specifically, we needed to consider a role for CCR2 inhibition in sensory neurons. A well-established downstream target of CCR2 signalling in neurons is p-ERK activation, which is associated with noxious activation of sensory neurons [3CR1-deficient and heterozygous mice. However, RS-102895 reduced VCR-associated p-ERK2 expression to the same extent in both genotypes despite the fact that allodynia is significantly reduced in CX3CR1-deficient mice. Although this does not rule out a role for CCR2-mediated neuronal activation in VCR allodynia, since thresholds observed in CX3CR1-deficient mice are still considered to be allodynic, it could suggest that other CCR2-mediated mechanisms are just as pivotal in regulating the severity of allodynia. Another key observation was that after the treatment was terminated, p-ERK2 activation in VCR-treated mice that also received RS-102895 was no longer reduced, regardless of the genotype. Crucially, however, allodynia was still significantly reduced in CX3CR1-deficient mice, despite measureable p-ERK2 activation. This could suggest one of two things: either the reduction in VCR allodynia in RS-102895-treated CX3CR1-deficient mice does not depend on the effect on neuronal activation or the dominant underlying mechanisms regulating VCR allodynia change as VCR pain evolves into ongoing after VCR treatment is terminated. Regardless of the explanation, however, our data suggests that there is scope for CCR2-mediated mechanisms that do not only require activation of CCR2 in sensory neurons to regulate VCR-associated allodynia in CX3CR1-deficient mice. CCR2 signalling in monocytes could be a candidate for such a mechanism.Whereas CX neurons . To attr neurons . At the 3CR1 in human monocytes in vitro regulated basal release of the proinflammatory cytokines TNFα and IL1β. It is well-established that both TNFα and IL1β are pronociceptive [3CR1-CCR2 interaction in monocytes and noxious signalling. Indeed, we confirmed that CCL2 stimulation of THP-1 cells also results in TNFα and IL1β release and observed that both CCL2 and CCR2 are elevated in THP-1 cells when CX3CR1 is downregulated thus suggesting that downregulation of CX3CR1 results in the release of proinflammatory cytokines through heightened CCL2/CCR2 signalling. Our data suggests the presence of a novel interaction between CX3CR1 and CCR2 receptors, which regulates the release of monocyte-derived signals that mediate communication between monocytes and nociceptive neurons


Thus, adding β-catenin antagonist (FH535 or DKK1) down-regulates β-catenin and c-myc expressions, and suppresses tumorigenesis  in hESC fed with MEF, while adding the β-catenin enhancer (LiCl or 6-bromoindirubin-3′-oxime) up-regulates the expressions, and has a trend (p = 0.056) to promote tumorigenesis (2/7 vs. 0/21) in hESC fed with HUCMSC. Furthermore, FH535 supplement does not alter the pluripotency of hESC when fed with MEF, as indicated by the differentiation capabilities of the three germ layers. Taken together, this investigation concludes that WNT/β-catenin/c-myc pathway causes the tumorigenesis of hESC on MEF feeder, and β-catenin antagonist may be adopted as a tumor suppressor.Human pluripotent stem cells harbor hope in regenerative medicine, but have limited application in treating clinical diseases due to teratoma formation. Our previous study has indicated that human umbilical cord mesenchymal stem cells (HUCMSC) can be adopted as non-teratogenenic feeders for human embryonic stem cells (hESC). This work describes the mechanism of non-tumorigenesis of that feeder system. In contrast with the mouse embryonic fibroblast (MEF) feeder, HUCMSC down-regulates the WNT/β-catenin/ Stem cell research has been widely performed in recent decades, and holds much hope for regenerative medicine. The most promising stem cells are pluripotent human embryonic stem cells (hESC) and human-induced pluripotent stem cells (iPSC)CMYC oncogene is downregulated in hESC with HUCMSC co-culture. This study assumes that HUCMSC may confer a signal that down-regulates CMYC in hESC.Human umbilical cord mesenchymal stem cells (HUCMSC) from epiblasts of the human embryo can be robustly isolated from the Wharton’s jelly of the umbilical cord after birth3453polyposis coli (APC), Axin and the kinases glycogen synthase kinase-3β (GSK-3β) and casein kinase I, marking it for ubiquitin-mediated proteolysis. Binding of Wnt to the cell surface Frizzled receptors and LRP5/6 co-receptors protects β-catenin from degradation, and acts on its targets, including c-myc, to promote cell cycle progression and inhibit apoptosis1314Myc is a downstream target of β-catenin91011c-Myc and the non-tumorigenic feature of hESC. We found that β-catenin signaling is the main factor controlling tumorigenesis, and that its inhibition mimics the tumor suppressor activity of HUCMSC.This investigation explores the signaling pathway responsible for the HUCMSC-mediated down-regulation of c-myc in the three types of co-culture feeders were first investigated. Shifting from the MEF feeder to the HUCMSC feeder reduced the expression of mRNA and protein of β-catenin in hESC. The expression of β-catenin rebounded after shifting back to the MEF feeder ; GATA4 (endoderm); GATA6, Hand1 (mesoderm) were not altered in hESC cultured on either MEf or HUCMSCs feeder  in hESC/HUCMSC, teratoma formed in 2/7 (28.6%) grafted mice, compared to 0/21 (0%) in the non-treated group  p = 0.0.c-myc signaling, the hESC/MEF were treated with the β-catenin signaling inhibitor FH535, and subjected to xenograft in NOD/SCID mice. After 3 months, FH535 significantly inhibited teratoma formation of hESC/MEF. Only 3 of 14 (21.4%) injection sites developed teratoma, compared to 4 of 4 (100%) in the non-treated group , endoderm (GATA4), mesoderm  and ectoderm  were noted with levels equivalent to those without FH535 treatment  to down-regulate survivin signalingThe non-tumorigenesis of HUCMSC coculture has many possible factors in the upstream signal of β-catenin. WIF (or sFRP) may be secreted to counteract the Wnt-Frizzled binding and down-regulate beta-catenin signaling4041c-myc signaling inhibits tumor formation. Inhibition of this signaling by β-catenin inhibitor could markedly reduce the incidence of teratoma formation in the conventional hESC/MEF co-culture system.In summary, this study reveals that the hESC/HUCMSC co-culture can confer a non-tumorigenesis phenotype of hESC. Down-regulation of the β-catenin/https://www.thermofisher.com/), 50 μM B-mercaptoethanol  and 4 ng/ml basic fibroblast growth factor (bFGF). The medium was changed daily, and the hESC was passed each week.The Research Ethics Committee of Buddhist Tzu Chi General Hospital (IRB 100–166) approved the protocols for collecting and using human umbilical cord. Written informed consent was obtained from the pregnant women before labor. The methods were performed in accordance with the relevant guidelines, including any relevant details. The TW1 hESC was obtained from the Food Industry Research and Development Institute of Taiwan, and maintained on the mitomycin-C treated MEF (hESC/MEF) or HUCMSC (hESC/HUCMSC) following a previously reported protocolTo prepare the hESCs with different feeders, hESC was first co-cultured with MEF (hESC/MEF), and the established hESC clusters were transferred to the HUCMSC feeder as the hESC/HUCMSC co-culture. For the reverse co-culture, hESC was further transferred to the MEF feeder as the hESC/MHM. All the feeder transfers were performed after five passages in the previous feeder. The hESC/MHM were also maintained for more than five passages before investigation.FH535, LiCl and BIO (6-bromoindirubin-3′-oxime) were purchased from Sigma-Aldrich. DKK1 was purchased from R & D systems.https://lifescience.roche.com) gene expression assays in an ABI Step One Plus system , with GAPDH as an internal control. RNA for all qRT-PCR and RT-PCR analysis was prepared using Trizol (Invitrogen) and quantified. 500ng of RNA was DNAase-treated using DNaseI amplification grade (Invitrogen). The first strand of cDNA was synthesized by a SuperScript III One-Step RT-PCR kit (Invitrogen) following the manufacturer’s instructions. All PCR samples were analyzed by electrophoresis on 2% agarose gel containing 0.5 μg/ml ethidium borome (Sigma). The quantitative RT-PCR (qRT-PCR) analysis adopted FastStart universal SYBR green master  was adopted for ChIP assay for beta-catenin-CMYC promoter binding. The assay with beta-catenin antibody was performed with 4 × 10http://www.gehealthcare.com/). The membranes were incubated with specific monoclonal antibodies. The secondary antibody was HRP-conjugated goat anti-mouse IgG . The bound antibodies were detected by enhanced chemiluminescence reagents .The cells were lysed in the lysis buffer  plus proteinase inhibitor cocktail (Roche). The proteins were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to a nitrocellulose membrane  was adopted to isolate protein from the nucleus and cytoplasm. The isolation procedures were conducted according to the manufacturer’s instructions. The resulted cytoplasmic/nuclear protein were analysed by Western blot.The TCF/LEF signal reporter assay kit (Qiagen) was adopted to demonstrate the transactivating activity of β-catenin. Briefly, one day before transfection, ES cells were seeded at a density of 30,000 cells per well into a 96-well plate in 100 μl of medium. Next day, 1 μl of TCF/LEF luciferase reporter (component A) was transfected from each well into cells. After 24 h of transfection, 55 μl of Luciferase reagent per well were added and shaken at room temperature for 15 min, and firefly luminescence was measured by a luminometer. 55 μl of Stop & Glo reagent per well were added and rocking at room temperature for 15 min, and Renilla luminescence was measured. To obtain the normalized luciferase activity for TCF/LEF reporter, the background luminescence was calculated as the ratio of firefly luminenscence from the TCF/LEF reporter to Renilla luminescence, and subtracted from the control Renilla luciferase vector. Graphs were plotted from data obtained as a mean of three independent experiments, with standard deviation shown as error bars.5 cells mixed with Matrigel (1:1) were injected into the back subcutaneous tissue of 6–8-week-old female non-obese diabetic-severe combined immunodeficiency (NOD-SCID) mice. Tumor formation was followed up by palpation. The resulting tumors were dissected, fixed, embedded in paraffin and processed for histological examination.All animal works were in accordance with protocols approved by the Institutional Animal Care and Use Committee at the Buddhist Tzu Chi General Hospital. All methods were performed in accordance with the relevant guidelines and regulations. For the tumorigenesis assay, hESC were removed from the feeder with mechanical slicing using glass capillaries, then pelleted and resuspended in PBS. For the xenograft, 5 × 10www.emdmillipore.com/).The hESC colonies were cultured on chamber slides  in culture dishes with feeder cells, then subjected to immunohistochemistry 3–7 days following passage. Cells were fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100, blocking with 4% normal goat serum, then treatment with primary antibodies such as Sox2, stage-specific embryonic antigen-4 (SSEA4), TRA-1-60 and TRA-1-81  formation was performed for 5 days. The resulting EB was plated on gelatin-treated chamber slides and fixation. Antibodies specific for three germ layers, namely ectoderm , mesoderm (brachyury) and endoderm [AT motifi-binding factor 1 (ATBF-1)], were identified.http://www.ibm.com/). Data are shown as mean ± SE. The means were compared by one-way ANOVA followed by Tukey correction. The level of statistical significance was set at p < 0.05.All analyses were conducted by the software IBM SPSS Statistics version 21 .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In a previous study, human bone marrow mesenchymal stem cells (hBMSCs) with reduced SIRT7 activity were developed to evaluate the effect of SIRT7 on osteogenesis. SIRT7 knockdown significantly enhanced osteoblast-specific gene expression, alkaline phosphatase activity, and mineral deposition in vitro. Additionally, SIRT7 knockdown upregulated β-catenin. The enhanced osteogenesis due to SIRT7 knockdown was partially rescued by a Wnt/β-catenin inhibitor. Furthermore, SIRT7 knockdown hBMSCs combined with a chitosan scaffold significantly promoted bone formation in a rat tibial defect model, as determined by imaging and histological examinations. These findings suggest that SIRT7 has an essential role in osteogenic differentiation of hBMSCs, partly by activation of the Wnt/β-catenin signaling pathway.Sirtuin 7 (SIRT7) is a NAD A better understanding of the osteogenesis of MSCs is necessary for clinical applications in bone regeneration.2 Thus discerning the genetic factors involved in MSCs osteogenesis is an important area of research.Bone marrow stem cells (BMSCs) possess self-renewal capabilities and the potential to differentiate into a variety of cell types, including osteoblasts, chondrocytes, and adipocytes.5 SIRT7 binds to the ribosomal RNA gene and is associated with reactivation of ribosomal DNA transcription following mitosis.6 Barber et al.7 revealed that SIRT7 is a NAD+-dependent H3K18Ac (acetylated lysine 18 of histone H3) deacetylase that stabilizes the transformed state of cancer cells. Moreover, several studies reported that SIRT7 is overexpressed in breast cancer tissues and in thyroid and colon tumors compared with normal tissue,9 and it showed a close relationship with inflammatory cardiomyopathy and apoptosis.10 In addition, sirtuin family genes and osteogenic differentiation are closely related. SIRT1 is a regulator of bone mass and a repressor of sclerostin.11 SIRT3 is required for osteogenesis via modulation of mitochondrial function and biogenesis via PGC-1α/SOD2 signaling.12 Sun et al.13 showed an essential role of SIRT6 in osteogenesis of rat MSCs partly by suppressing NF-κB signaling. However, little is known about the role of SIRT7 in the osteogenesis of MSCs.SIRT7 is a member of the sirtuin family of histone deacetylases, the members of which have diverse roles in aging, DNA repair, cell cycle, metabolism, and disease biology.β-catenin signaling is a crucial regulator of MSCs that has important roles in osteogenic differentiation.15 The canonical Wnt signaling pathway is activated upon Wnt binding to frizzled receptors16 and LRP co-receptor at the cell membrane,18 causing inhibition of glycogen synthase kinase-3β (GSK3β) and stabilizing cytoplasmic β-catenin.15 Via activation of Wnt, β-catenin accumulates in the cytosol and translocates to the nucleus where it promotes T-cell factor/lymphoid enhancing factor-1-mediated transcription,20 thereby affecting target gene transcription. Moreover, several secreted molecules are antagonists of Wnt signaling, including secreted glycoproteins dickkopfs (Dkks) and secreted frizzled-related proteins.21 Of these, Dkk1 was shown to inhibit the canonical Wnt signaling pathway in several different cell types.25 Previous studies have shown that Wnt signaling is critical for bone formation and maintenance and that canonical signaling promotes stromal progenitor proliferation and osteogenesis.28 Additionally, the sirtuin family and Wnt signaling are closely related. SIRT1 promotes bone formation by preventing β-catenin sequestration by FoxO transcription factors.29 As a tumor suppressor, SIRT2 inhibits the Wnt signaling pathway by binding to β-catenin produced in response to radiation-induced stress.30 Wang et al.31 found that SIRT6 regulates hematopoietic stem cell homeostasis and self-renewal capacity via Wnt signaling.Wnt/β-catenin signaling. By assessing the expression levels of specific osteogenic markers and calcium deposition, we revealed that SIRT7 knockdown enhances osteogenic differentiation of hBMSCs partly via the Wnt/β-catenin signaling pathway in vitro. We also used a rat tibial defect model transplanted with SIRT7 knockdown hBMSCs, combined with the use of a chitosan scaffold, and found that SIRT7 knockdown promoted healing of bone defects in vivo.In this study, we investigated the expression of SIRT7 in human BMSCs (hBMSCs) and its effect on the osteogenic differentiation of hBMSCs. We found that endogenous expression of SIRT7 decreased during osteogenic differentiation. We hypothesized that downregulation of SIRT7 promotes osteogenesis of hBMSCs via Wnt/To determine the expression levels of SIRT7 associated with osteogenic differentiation of MSCs, we examined SIRT7 endogenous expression in hBMSCs at days 0, 3, and 7 during osteogenic differentiation. Compared with undifferentiated hBMSCs, both the mRNA  and protTo clarify the role of SIRT7 in osteogenic differentiation, a lentiviral vector system was used to efficiently knock down SIRT7 of third-generation hBMSCs. SIRT7 knockdown was quantified by evaluating the ratio of green fluorescent protein (GFP)-positive cells to the total cell number . SIRT7 eTo determine whether SIRT7 knockdown influences the proliferation of hBMSCs, we measured CCK8 levels in hBMSCs. The effects of SIRT7 knockdown on hBMSC proliferation at days 1, 3, and 5 after infection are shown in P<0.05, To evaluate the role of SIRT7 knockdown in osteogenic differentiation, the levels of osteo-specific genes and proteins, including runt-related transcription factor 2 (RUNX2), osterix (OSX), osteopontin (OPN), and collagen type I alpha 1 chain (COL1A1), were determined by quantitative real-time PCR (qPCR) and western blotting analyses. qPCR analysis revealed that RUNX2, OSX, OPN, and COL1A1 mRNA levels were significantly higher in SIRT7 knockdown hBMSCs than in the control group at days 3 and 7 P<0.05, . WesternWe also performed an immunofluorescence analysis to assess RUNX2 protein expression, and the results showed that RUNX2 expression levels were increased on day 3 in SIRT7 knockdown cells . SIRT7 pP<0.05, ALP activity is a marker of early-stage osteogenesis. We evaluated ALP activity at days 3 and 7 during osteogenic differentiation. Compared with the control group, higher ALP activity was observed in the SIRT7 knockdown group P<0.05, . Similarβ-catenin signaling, the expression of β-catenin was determined by qPCR, western blotting analysis, and immunofluorescence analysis during osteogenesis at days 3 and 7. The expression of GSK3β and Axin was also determined by qPCR. The results of the qPCR and western blotting analyses revealed higher expression of β-catenin in the SIRT7 knockdown hBMSCs  β-catenin was significantly decreased compared with the level in SIRT7 knockdown hBMSCs without the inhibitor  also indicated Sir2 homologs as determinative factors of lifespan.34 Prokaryotic and eukaryotic sirtuins are homologous with yeast Sir2, and seven mammalian homologs have been identified so far.35 Among these, three mammalian sirtuins  are located in the nucleus, with SIRT1 also found in the cytoplasm. SIRT7 is located in the nucleolus in human cells and associates with ribosomal DNA and interacts with RNA polymerase I.35 Ford et al.36 found that SIRT7 activity requires the amino-acid residues that bind to NAD within the conserved sirtuin core domain, indicating a role for NAD-dependent regulation. SIRT7-deficient mice experience a reduction in lifespan and progressive heart hypertrophy and inflammatory cardiomyopathy.10 Overexpression of SIRT7 exhibits oncogenic properties, such as promotion of colony formation, a more invasive phenotype, and cell growth both in vitro and in vivo.37 A recent report showed that SIRT7 acted as an oncogene in gastric cancer and hepatocellular carcinoma.39 Recently, SIRT7, the least-investigated member of the sirtuin family, has been a focus of research of diseases, such as hypertrophic inflammatory cardiomyopathy, fatty liver disease, oncogenic transformation, tumor growth, and age-related hearing loss.Yeast silent information regulator 2 (Sir2), a member of the sirtuin family, was originally identified in a screening for silencing factors.et al.36 The effect of SIRT7 in hBMSCs during osteogenesis was evaluated by qPCR and western blotting analyses, which showed endogenous SIRT7 expression to be significantly downregulated at days 3 and 7 after osteogenic differentiation. We constructed SIRT7 knockdown hBMSCs for additional experiments. ALP staining and calcium deposits are regarded as early and late markers of osteoblastic differentiation, respectively.40 We found that SIRT7 knockdown enhanced ALP activity and accelerated mineralization. RUNX2, a master transcription factor in osteogenic differentiation,41 was significantly increased in expression following knockdown of SIRT7, as determined by qPCR, western blotting, and immunofluorescence analysis. Other osteogenic markers, such as OSX, Col1, and OPN, showed similar patterns. Meanwhile, SIRT7 knockdown did not affect proliferation of hBMSCs. These results suggest that SIRT7 knockdown promotes osteogenesis of hBMSCs in vitro.Immunofluorescence analysis showed that SIRT7 is located in the nucleus, consistent with the observation of Ford β-catenin signaling is an essential signaling pathway required for bone formation.42 Under the influence of the Wnt ligand, frizzled and LRP-5/6 co-receptors recruit Disheveled to the plasma membrane together with Axin-GSK3β, inhibiting formation of the β-catenin complex and increasing β-catenin levels in the cytoplasm.44 This leads to nuclear translocation of β-catenin and activation of target genes.45 In our present study, we observed that SIRT7 knockdown increased the expression of β-catenin during osteogenesis, and immunofluorescence analysis confirmed these observations. Furthermore, the increased osteogenesis of hBMSCs by SIRT7 knockdown was partially rescued by an inhibitor of Wnt/β-catenin (DKK1). Knockdown of β-catenin by RNA interference using small interfering RNAs produced similar results. These findings indicate that knockdown of SIRT7 regulates osteogenic differentiation of hBMSCs via activation of the Wnt/β-catenin signaling pathway.Wnt/47 Polyampholytic chitosan fibers promoted proliferation and osteogenic differentiation of MSCs, as well as osseous tissue regeneration, in a rabbit model.48 In our study, the use of a porous chitosan scaffold with hBMSCs promoted bone healing in a rat tibial defect model. Better bone formation was observed when SIRT7 knockdown hBMSCs were present in the scaffold.A previous report demonstrated that porous chitosan-alginate scaffolds are osteoconductive, and experimental treatments showed improved defect closure in a calvarial defect model.β-catenin signaling pathway by SIRT7 knockdown are not fully clarified, especially with regard to the nuclear translocation of β-catenin. Other signaling pathways need to be examined for potential involvement in the osteogenesis of hMSCs by SIRT7 knockdown in future studies.Many studies have demonstrated a relationship between expression of sirtuins and stem cell osteogenesis. However, this is the first study to demonstrate the impact of SIRT7 on osteogenic differentiation of MSCs. Unfortunately, we determined the effect of only SIRT7 knockdown, and not SIRT7 overexpression, on osteogenesis. Moreover, the mechanisms of activation of the Wnt/β-catenin signaling pathway. SIRT7 knockdown in hBMSCs combined with a chitosan scaffold enhanced bone defect repairs and may provide a new stem cell-based strategy for bone regeneration.According to our results, SIRT7 knockdown enhanced osteogenic differentiation of hBMSCs, partly via activation of the Wnt/2 and were passaged after reaching 80% confluence. Cells from passages 3–9 were used in subsequent experiments. Recombinant DKK1 was purchased from PeproTech . Based on a previous study, we used a DKK1 of 0.5 μg/ml.49hBMSCs, purchased from Cyagen Biosciences , can differentiate into osteoblasts, adipocytes, and chondrocytes under specific inductive conditions. Adherent hBMSCs were cultured in culture flasks in hMSC growth medium  in an incubator at 37 °C with 5% COμg/ml polybrene in growth medium at a multiplicity of infection of 50. After 12 h, >95% of the cells were still viable, and the culture medium was then changed. Three days later, all transfected cells were passaged for use in subsequent experiments. The expression of SIRT7 was determined by qPCR and western blotting analyses.Lentiviral knockdown SIRT7 (lenti-SIRT7) particles and lentiviral GFP particles, used as the control group (lenti-control), were prepared by GenePharma Co., Ltd . For infections, 40–60% confluent hBMSCs were incubated with lentiviral particles and 2.5 μl low-sugar Dulbecco’s modified Eagle’s medium (L-DMEM) without fetal bovine serum (FBS) for 3 h at 37 °C. Absorbance at 450 nm, which is directly proportional to cell proliferation, was measured using a microplate reader .To assess the effect of SIRT7 knockdown on the proliferation of hBMSCs, cells were seeded into a 96-well plate (5000/well) and allowed to adhere for 24 h. After 24 h, the medium was removed, and the cells were treated with 10% Cell Counting Kit-8  in 100 4/cm2 and incubated for 48 h at 37 °C under 5% CO2. The cells were subsequently cultured in osteogenic induction medium . The cells were maintained by the addition of fresh osteogenic induction medium every 2–3 days.hMSCs were cultured in growth medium  and 100 IU/ml penicillin/streptomycin) in 6- or 12-well cell culture plates at a density of 3 × 10Cells were cultured in osteogenic induction medium in 12-well plates for 3 or 7 days. For ALP staining, cells were fixed with 4% paraformaldehyde for 15 min. Cells were then washed twice with PBS and stained using the BCIP/NBT Alkaline Phosphatase Color Development Kit . For measurement of ALP activity, cells were lysed with lysis buffer consisting of 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 1% Triton X-100. ALP activity was determined using the ALP Activity Assay (Beyotime) according to the manufacturer’s instructions. Briefly, the conversion of colorless p-nitrophenyl phosphate to colored p-nitrophenol was measured after 3 and 7 days of culture in osteogenic medium at 405/650 nm.μl were plated on 96-well plates, which were read at 560 nm using a microplate reader . The readings were normalized to the total protein concentration.After induction of osteogenic differentiation, mineral deposition was assessed by ARS (Cyagen Biosciences). Cells were fixed in 4% paraformaldehyde for 15 min at room temperature and subsequently washed with distilled water. The cells were incubated with a 0.5% solution of alizarin red for 20–30 min at room temperature, followed by rinsing with distilled water. The stain was desorbed by incubating with 10% cetylpyridinium chloride  for 1 h. The solution was collected, and 200 μl using the Double-Strand cDNA Synthesis Kit (Takara). One microliter of cDNA was used as the template for qPCR. All gene transcripts were quantified by qPCR using the Power SYBR Green PCR Master Mix (Takara) on the ABI StepOnePlus System . The mRNAs of the target genes and the housekeeping gene (GAPDH) were quantified in separate tubes. All primers were synthesized by Sangon Biotech . The primer sequences used are shown in −ΔΔCt method.Total cellular RNA was isolated using RNAiso reagent  and quantified by measuring the absorbance at 260 nm . First-strand cDNA was synthesized using PrimeScript RT Master Mix (Takara) according to the manufacturer’s instructions. Total RNA (⩽1000 ng) was reverse-transcribed into cDNA in a reaction volume of 20 μg/ml; Abcam, Shanghai, China), RUNX2 , COL1A1 , non-phosphorylated (active) β-catenin , or total β-catenin . After washing in TBST four times (5 min each), the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies  for 1 h at room temperature. After washing five times with TBST, we detected proteins using enhanced chemiluminescence blotting reagents according to the manufacturer’s instructions. The immunoreactive bands were detected using an enhanced chemiluminescent detection reagent (Millipore). Signal intensity was measured using the Bio-Rad XRS chemiluminescence detection system .Cells were lysed in RIPA buffer supplemented with a proteasome inhibitor (Beyotime). Equal amounts of proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane . After blocking in 5% non-fat milk for 2 h, the membranes were incubated overnight at 4 °C with antibodies specific to GAPDH , SIRT7  as follows. Cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized, and blocked for 30 min in 0.05% Triton X-100 and 2% bovine serum albumin. Fixed cells were washed and incubated overnight with anti-RUNX2 , SIRT7 , or non-phosphorylated (active) β-catenin . Cells were incubated with a fluorescence-conjugated secondary antibody (Beyotime) for 120 min, and nuclei were stained with 4′,6-diamidino-2-phenylindole  for 5 min. Samples were observed under a fluorescence microscope (Leica).Cells were cultured in induction medium in a 12-well plate and evaluated for RUNX2, SIRT7, and 2) were placed in 24-well plates. Once placed into sterile tissue culture plates, scaffolds were incubated for 4 h in growth medium to remove any residual alcohol and to wet the scaffolds. Cells were seeded at a density of 50 000/scaffold in 50 μl medium and incubated at 37 °C for 3 h to ensure attachment; the medium was then brought to 600 μl/well. Medium was exchanged after 24 h and changed every day.Chitosan scaffolds, which were gifts from Professor Hu, were sterilized by incubation in 70% ethanol for 30 min followed by UV exposure for 30 min on each side. The chitosan scaffolds (1 × 1 cmAll Sprague Dawley (SD) rats were supplied by the Academy of Medical Sciences of Zhejiang Province. All animal experiments were performed in accordance with the Animal Care and Use Committee guidelines of Zhejiang Province. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Zhejiang University.51 A 1.5-mm-diameter tibial defect was made in all SD rats approximately 5 mm from the proximal tibial growth plate by a hollow drill. The same leg was used for each group. The 24 defects in 24 rats were randomized into four groups. In the blank group (n=6), nothing was grafted onto the fracture site; in the negative-control group (n=6), the defect areas were filled with chitosan scaffold only; in the lenti-control group (n=6), the scaffolds seeded with lenti-control hBMSCs were implanted into the tibial defects; and in the lenti-SIRT7 group (n=6), the scaffolds with lenti-SIRT7 hBMSCs were implanted into the defects.All surgical procedures were performed by two experienced traumatic orthopedic surgeons (ZJP and DTX). Tibial defects were generated in 8-week-old male SD rats (weighing approximately 200 g). The rats were anesthetized intraperitoneally with 0.3% pentobarbital sodium (Sigma) at 30 mg/kg body weight. After anesthesia, an incision was made below the knee. An intramedullary needle (1.2-mm-diameter stainless steel syringe needle) was inserted inside the medullary canal of the tibia for fixation. The tibial defect model was established as reported previously.52Animals were killed 6 weeks after surgery, and the samples were collected. Radiographs were taken using a dual-track molybdenum/rhodium+ Mo target mammography machine  to evaluate callus formation at the defect site. All samples were scanned for bone formation using a micro-CT 100 imaging system  with the following scan parameters: 70 kVp X-ray energy, 1024 reconstruction matrix, 0.0148 mm slice thickness, and 300 ms integration time. BV/TV, Tb.N, and Conn.D were calculated by three-dimensional standard microstructural analysis.μm thickness were cut and mounted on polylysine-coated slides. Hematoxylin and eosin, Safranin O and Fast green, and Masson staining were performed separately on consecutive tissue sections. For hematoxylin and eosin staining, tissue sections after deparaffinization were rehydrated and stained with hematoxylin for 30 s, rinsed in water for 1 min, eosin for 10–30 s, and dehydrated with alcohol. For Safranin O and Fast green staining, tissue sections after deparaffinization were rehydrated and stained with hematoxylin for 30 s, Fast green for 15 min, rinsed in PBS for 15 min, Safranin O for 5 min, and dehydrated with alcohol. For Masson staining, tissue sections after deparaffinization were rehydrated and stained with hematoxylin for 30 s, Biebrich scarlet-acid fuchsin for 5 min, rinsed in PBS for 15 min, phosphomolybdic acid for 5 min, rinsed in PBS for 15 min, Aniline blue for 5 min, and dehydrated with alcohol. Images were obtained using a microscope.The samples were fixed in 4% paraformaldehyde for 72 h at room temperature and then decalcified using 10% EDTA (Sigma), changing the solution once a week for >8 weeks, before embedding in paraffin. Serial sections of 3-Statistical analysis was performed using the SPSS 17.0 software . All experiments were performed in at least triplicate, and the data are presented as means±S.D.t-test when comparing two groups and one-way ANOVA followed by Bonferroni’s post hoc test when comparing more than two groups. A P-value⩽0.05 was considered to represent a statistically significant difference.Statistical significance was determined using a two-tailed Student’s Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.