Wnt/β-catenin signaling, a highly conserved pathway through evolution, regulates key cellular functions including proliferation, differentiation, migration, genetic stability, apoptosis, and stem cell renewal. The Wnt pathway mediates biological processes by a canonical or noncanonical pathway, depending on the involvement of β-catenin in signal transduction. β-catenin is a core component of the cadherin protein complex, whose stabilization is essential for the activation of Wnt/β-catenin signaling. As multiple aberrations in this pathway occur in numerous cancers, WNT-directed therapy represents an area of significant developmental therapeutics focus. The recently described role of Wnt/β-catenin pathway in regulating immune cell infiltration of the tumor microenvironment renewed the interest, given its potential impact on responses to immunotherapy treatments. This article summarizes the role of Wnt/β-catenin pathway in cancer and ongoing therapeutic strategies involving this pathway. INT1. Simultaneous work in developmental biology and work in drosophila established INT1 gene to be the homologue of the Drosophila segment polarity gene, Wingless , 8685, 8+ T cell infiltration and priming, therefore possibly producing a more favorable scenario to immune checkpoint inhibition [These results suggest that increased expression of β-catenin in the tumor should be investigated and validated as a predictive tool to improve the selection of candidate patients for checkpoint inhibition therapy. In addition, one might hypothesize that inhibition of Wnt/β-catenin signaling could improve CD8hibition . Thus, t+ effector T cell is favorable, presence of regulatory T cells (Treg) has been associated with poor antitumor immunity. Tregs are known to suppress adaptive responses mainly by reducing CD8+ T cell proliferation, activation, and effector function [+ T cells using lentiviral transduction. This led to increased IL-17a expression, enhanced proliferation, and inhibited apoptosis of colorectal cancer cells [+ T cells by blocking T cell differentiation [While tumor infiltration with CD8function , 91. Somfunction , 93. In er cells . Active er cells . Hence, ntiation . This stntiation .CCL4. The defective production of CCL4 led to impaired infiltration and activation of Batf3-lineage CD103+ dendritic cells, reduced CD8+ T cell priming and infiltration, and consequent non-response to immune check point blockade. In the absence of active β-catenin signaling, the normal production of CCL4 was restored, leading to CD103+ dendritic cell activation and infiltration and proficient priming of CD8+ T cell. Wnt/β-catenin signaling in intestinal dendritic cells has shown to regulate the balance between inflammatory versus regulatory responses in the gut [Wnt/β-catenin pathway has also been associated with modulation of innate immunity, such as dendritic cells . Sprange the gut . β-caten the gut .Wnt/Beta-catenin signaling has been shown to be involved in cross talk between cancer cells and tumor-associated macrophages. Kaler et al. demonstrated a possible role of interleukin-1β secreted by TAMs in the increased availability of β-catenin through phosphorylation of GSK3β in colon cancer cells, disrupting the function of β-catenin destruction complex . ColorecThe Wnt/β-catenin pathway is increasingly recognized as a potentially important target for anticancer therapies, with several relevant inhibitors at various stages of clinical development. The expanding role of immunotherapies in cancer and recent insight into the role of Wnt-pathway in cancer-related immune-regulation may give a new dimension to this field of developmental therapeutics. Only tumors with upregulated Wnt/β-catenin signaling such as colorectal cancer have been explored as targets for Wnt inhibition. However, with its role in immunomodulation, Wnt inhibitors may have a broader role in cancers such as melanoma, lung, and renal cancers where immunotherapy has come to the forefront. There are some nuances to be addressed in the exact role of Wnt signaling in immunomodulation before this could be adopted in clinical practice.


LncFZD6 drives liver TIC self-renewal and tumor initiation capacity through FZD6-dependent manner. LncFZD6 interacts with BRG1-embedded SWI/SNF complex and recruits it to FZD6 promoter, and thus drives the transcriptional initiation of FZD6 by chromatin remodeling. WNT5A, a ligand of FZD6, is highly expressed in liver non-TICs and drives the self-renewal of liver TICs through lncFZD6-BRG1-FZD6-dependent manner. Through FZD6 transcriptional regulation in cis, lncFZD6 activates Wnt/β-catenin signaling in liver TICs. LncFZD6-BRG1-Wnt5A/β-catenin pathway can serve as a target for liver TIC elimination. Altogether, lncFZD6 promotes Wnt/β-catenin activation and liver TIC self-renewal through BRG1-dependent FZD6 expression.Liver tumor-initiating cells (TICs), the drivers for liver tumorigenesis, accounts for liver tumor initiation, metastasis, drug resistance and relapse. Wnt/β-catenin signaling pathway emerges as a critical modulator in liver TIC self-renewal. However, the molecular mechanism of Wnt/β-catenin initiation in liver tumorigenesis and liver TICs is still elusive. Here, we examined the expression pattern of 10 Wnt receptors (FZD1–FZD10), and found only FZD6 is overexpressed along with liver tumorigenesis. What’s more, a divergent lncRNA of FZD6, termed  Liver cancer is one of the most serious cancers in the world. Liver cancer contains two common tumor types, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). Heterogeneity is a major characteristic of liver cancer and largely increases the difficulty for clinical elimination . ActuallSeveral signaling pathways participate in liver TIC self-renewal, including Wnt/β-catenin signaling, Notch signaling and Hedgehog signaling –15. AmonFZD receptors, encoded by “frizzled” gene family, are the receptors for Wnt molecules, and responsible for Wnt/β-catenin activation upon Wnt binding . There alncFZD6 and FZD6 are highly expressed in liver cancer cells and liver TICs. LncFZD6 and FZD6 are required for liver TIC self-renewal. LncFZD6 interacts and recruits BRG1 to FZD6 promoter to initiate transcription. LncFZD6-BRG1-FZD6 signaling pathway can be a target for liver TIC elimination.Long noncoding RNAs (LncRNAs) are RNA transcripts with longer than 200 nucleotides in length and no protein-coding potential . Recent lncSox4 participated in liver TIC self-renewal and liver tumorigenesis. LncSox4 recruits Stat3 to the promoter of Sox4, and thus drives Sox4 expression and liver TIC self-renewal [Liver TICs account for liver initiation, invasion, metastasis and relapse. The self-renewal capacity of liver TIC self-renewal is under precise regulation. Using unbiased screening for lncRNAs, we found that a lncRNA termed -renewal . Wnt/β-cThe activation of Wnt/β-catenin needs the combination of Wnt ligands and Wnt receptors. The expression levels of Wnt receptors play a fundamental role in Wnt/β-catenin activation. Here, we examined the expression levels of Wnt receptor (FZD1–FZD10) using online available dataset (GSE14520) , 36. Of + liver TICs and oncospheres, FZD6 is highly expressed  is a divergent lncRNA of FZD6. The distance of their transcription start sites is <1 kb  . The staining results showed increased lncFZD6 expression levels in liver tumor, especially in advanced liver tumor , and found knockdown of these genes had no changes of  kb Fig. . LncFZD6les Fig. . Interes87) Fig. . We thenmor Fig. . Of noteles Fig. .Fig. 2LnlncFZD6 expression pattern, we next examined the expression of lncFZD6 in liver TICs. We obtained CD133+ liver TICs and oncospheres, examined lncFZD6 expression, and found increased lncFZD6 transcripts in liver TICs  was identified as the binding site with BRG1   Fig. . Finallyres Fig. . AltogetlncFZD6 and BRG1 in FZD6 transcription regulation. First, we performed chromosome immunoprecipitation (ChIP) and Chromatin isolation by RNA purification (ChIRP) assays, followed by FZD6 promoter examination, and found both BRG1 and lncFZD6 bound to FZD6 promoter. Of note, BRG1 and lncFZD6 interacted with the same region of FZD6 promoter  and HCC cell line PLC (ATCC CRL-8024) were obtained from ATCC. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium (GIBCO), supplemented with 100 μg/ml penicillin, 100 U streptomycin and 15% fetal bovine serum .Human liver cancer clinical samples were obtained from the first affiliated hospital of Zhengzhou University with informed consent, according to the Institutional Review Board approval. All human sample and mouse experiments were approved by the Institutional Committee of Zhengzhou University. HCC samples were ranked according to the obtained time, and several samples with sphere formation capacity  were used for sphere formation. The details for these sample were: #1, advanced HCC, 58 years old, male, tumor size, 7.8 × 5.2 × 4.9 mm, non-metastasis; #3, advanced HCC, 71 years old, male, tumor size, 8.2 × 4.3 × 3.2 mm, non-metastasis; #4, advanced HCC, 65 years old, female, tumor size, 5.8 × 5.2 × 4.6 mm, non-metastasis.DAPI (cat. no. 28718-90-3) and anti-β-actin antibody (cat. no. A1978) were purchased from Sigma-Aldrich. Anti-FZD6 (cat. no. HPA017991) antibody was from Atlas antibodies, anti-RNA polymerase II (cat. no. GWB-3F12B0) antibody was from GenWay. Anti-Oct4 (cat. no. 2750), anti-HeK4me3 (cat. no. 9727), anti-H3K27Ac (cat. no. 8173) antibodies were from Cell Signaling Technology. Anti-BRG1 (sc-17796) antibody was purchased from Santa Cruz Biotechnology. Phycoerythrin (PE)-conjugated CD133 (cat. no. 130098826) was from MiltenyiBiotec. Alexa594-conjugated donkey anti-rabbit IgG and Alexa488-conjugated donkey anti-mouse IgG antibodies were from Molecular Probes. T7 RNA polymerase (cat. no. 10881767001) and Biotin RNA Labeling Mix (cat. no. 11685597910) were obtained from Roche Life Science. The LightShift Chemiluminescent RNA EMSA kit (cat. no. 20158) and Chemiluminescent Nucleic Acid Detection Module (cat. no. 89880) were purchased from Thermo Scientific.For sphere formation assay, 5000 primary HCC cells were seeded into Ultra Low Attachment six-well plates and cultured in DMEM/F12 medium supplemented with N2, B27, 20 ng/ml EGF and 20 ng/ml bFGF for 2 weeks’ incubation. The materials needed for sphere formation were: ultra low attachment plates ; N2 supplement , B27 , epidermal growth factor (EGF)  and basic fibroblast growth factor (bFGF) .2O and used as template for reverse transcription-PCR (RT-PCR). RT-PCR was performed with RT-PCR kit derived from Promega Company, and real-time PCR was performed by standard procedures.Tissue RNA was extracted by TRIZOL method according to the Life’s manual. Briefly, 1 ml TriZol reagent was added into samples for 5-min incubation, followed by addition of 200 μl chloroform and then the samples were separated by centrifugation. The supernatant was incubated with isopropanol and washed with 75% ethanol. Finally, the RNA samples were dissolved with RNase-free HHCC samples or oncospheres were crushed within RIPA buffer , 1 mM EDTA, 1% NP-40 and 50 mM Tris, pH 8.0), followed by SDS–polyacrylamide gel electrophoresis (PAGE) for separation. The samples in the polyacrylamide gel were transferred to nitrocellulose (NC) membrane (Beyotime Biotechnology), and then incubated with primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies.2O2), the slides were boiled in Tris-EDTA buffer  for antigen retrieval. Then, the samples were incubated in primary antibodies and HRP-conjugated secondary antibodies. After detection with HRP substrate, the samples were counterstained with hematoxylin, followed by dehydration in graded alcohols and xylene.In all, 5-μm formalin-fixed sections were deparaffinized and rehydrated with xylene and graded alcohols. After 15-min incubation with 3% hydrogen peroxide . Then, the labeled RNA transcript was incubated with sphere lysate. Streptavidin beads were added and the enriched components were analyzed by SDS–PAGE and western blot or mass spectra.For RNA pulldown assays, biotin-labeled LncFZD6 or control ACTB enrichment was detected using real-time PCR.For RIP, liver TIC oncospheres were treated with 1% formaldehyde for crosslinking, and then lyzed with RNase-free RIPA buffer supplemented. The supernatants were incubated with BRG1 or control IgG antibodies and total RNA was extracted from the eluent. t-tests were used for statistical analysis. P < 0.05 was considered to be statistically significant.Two-tailed Student’s Supplementary Figure 1


There have been many attempts to unveil the therapeutic potential of antisense molecules during the last decade. Due to its specific role in canonical Wnt signalling, β-catenin is a potential target for an antisense-based antitumour therapy. In order to establish such a strategy with peptide nucleic acids, we developed a reporter assay for quantification of antisense effects. The luciferase-based assay detects splice blocking with high sensitivity. Using this assay, we show that the splice donor of exon 13 of β-catenin is particularly suitable for an antisense strategy, as it results in a truncated protein which lacks transactivating functions. Since the truncated proteins retain the interactions with Tcf/Lef proteins, they act in a dominant negative fashion competing with wild-type proteins and thus blocking the transcriptional activity of β-catenin. Furthermore, we show that the truncation does not interfere with binding of cadherin and α-catenin, both essential for its function in cell adhesion. Therefore, the antisense strategy blocks Wnt signalling with high efficiency but retains other important functions of β-catenin.The online version of this article (10.1007/s12033-018-0058-7) contains supplementary material, which is available to authorized users. In 1978 a synthetic antisense oligonucleotide was successfully used by Zamecnik and Stephensen for targeting of Rous sarcoma virus RNA within chick embryo fibroblasts , peakingDue to their good affinity for complementary RNA sequences, lack of interactions with natural proteins and excellent water solubility, morpholino oligos became the gold standard for antisense knock-down experiments , 8. TheyIn year 1991 peptide nucleic acids (PNAs) were developed by Nielsen and colleagues . Lack ofThe evolutionary highly conserved canonical Wnt pathway has shown to be crucial in development and disease . WithoutSeveral components of the Wnt pathway, including APC, Axin, Tcf and β-catenin, have shown mutations relevant for human cancers , 22. β-cIn this study, we show a newly designed splice-based reporter assay for quantification of antisense effects, which allows a positive luminescence read out for successful splice blocking by antisense oligos. Based on this assay, a dominant negative knock-down strategy was developed for β-catenin with specific PNA and morpholino oligos. For this purpose, β-catenin was targeted in a way that the interaction with its main partners Tcf/Lef is still possible; however, interaction with essential coactivators is blocked.For the splice-based reporter assay, a backbone containing a human EF1α short promoter  driving 2. Stable cell lines were generated using the piggybac transposon system [P values were calculated by the Student’s t test. Statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.HeLa, HEK293 T-REx (Invitrogen) as well as SW480 cells were cultured in Dulbecco’s modified Eagle’s medium  including 10% foetal bovine serum (Thermo Scientific) and 1% penicillin/streptomycin (Thermo Scientific) in a humidified environment with 5% COn system . PositivThe synthesis of the lysine–phosphonic–ester-modified PNAs was performed as described in Jung et al.  by ugich4 cells/well and grown over night (HeLa) or 48 h (HEK293 T-REx). Transfection was performed using TurboFect (Thermo Scientific) transfection reagent following instructions of the manufacturer. Measurement was performed as described [For transient transfection cells were seeded in a PEI-coated  96-well escribed . For the5 cells/well and incubated overnight. 24 h later medium was removed and 30 µL loading solution [Cells were seeded in a 24-well plate with 0.3 × 10solution  includin7 cells/mL and 30 µL transferred to a 1 mm gap electroporation cuvette (VWR) pre-cooled on ice. Adjustments for electroporation  on the For RNA extraction the GeneJET RNA Purification Kit (Thermo Scientific) was used following the instructions of the manufacturer. DNAse I (Thermo Scientific), random hexamer primers (Thermo Scientific) and RevertAid Reverse Transcriptase (Thermo Scientific) for cDNA synthesis were used according to the instructions of the manufacturer.4)2SO4, 0.02% Tween20, 0.2 µM dNTP mix (Thermo Scientific), 0.005 U Taq polymerase (Agrobiogen) and 1 µL cDNA were used. Additionally, for qPCR 1 µL SYBR green to a final dilution of 1:10 (Sigma), 20 µg bovine serum albumin and 4 mM MgCl2 or for Taqman 0.15 µM Taqman probes and 3.5 mM MgCl2 were added. Measurements were taken in a Stratagene MX3000P (Agilent Technologies).Primer and probe sequences are listed in Table S2. For each reaction 25 µL containing 0.2 µM of each primer, 80 mM Tris, 20 mM . 5 µg of the protein extracts (determined by Bradford assay) was run on an SDS page followed by semidry blotting. Membranes were blocked 1–2 h in 5% milk powder (Roth) in 1× TBS-T  on a shaker. Primary antibodies were diluted 1:500  or 1:10,000  and incubated O/N at 4 °C shaking. Secondary antibody  was added in a 1:5000 dilution with 5% BSA in TBS-T. Signals were detected with ECL solution (Santa Cruz Biotechnology) and a ChemiDoc (Protein Simple) station, and quantification was done with ImageJ.For development of an antisense strategy targeting β-catenin, we decided to apply a splice blocking approach , 43. In In order to develop a strategy for targeting of β-catenin, we first tested multiple PNAs directed against the SD regions of various exons and selected the exon 13 SD for further experiments Fig. a. Similap = 0.0011). Contrary to the luciferase-based assay, PNAs 4, 15 and 19 did not generate a significant reduction in the qPCR assay, whereas a morpholino oligo (MO3) directed to this SD resulted in a reduction of correctly spliced β-catenin mRNA to 59% compared to the mock-treated reference (Table S3). To demonstrate that the reduced detection of β-catenin mRNA was caused by aberrant splicing, an RT-PCR experiment was performed in SW480 cells . PNA18 showed significant effects also in this assay . On the other hand, a qPCR-based assay showed a significant reduction of β-catenin mRNA only for PNA18 , whereas non-significant values were obtained for the other 3 PNAs (p ≥ 0.3). Main reason for this discrepancy is the error rates, which are considerably smaller for the luciferase assay  are sufficient for selective binding. The small size makes them ideal for therapeutic applications, and the intracellular delivery can be further boosted using cell penetrating peptides , nanopar–18 basesDue to its relevance in cancer development, β-catenin has become a favoured target for nouveau cancer therapy strategies. Recent publications show decreased tumour burden within varying cancer types after targeting β-catenin with different approaches. PNAs have already been used for β-catenin knock-down in liver tumour cells  or antisAntisense-mediated splice blocking as a strategy to reduce β-catenin levels in the cell worked in our approach and resulted in reduced levels of β-catenin mRNA, protein and consequently also of target genes Fig. . A speciHerein, we present a splice-based reporter assay for accurate quantification of knock-down effects of antisense molecules. Furthermore, a strategy for targeting β-catenin next to its C-terminal transactivation domain resulted in a dominant negative protein, which efficiently blocks Wnt signalling, but retains interactions with cell adhesion molecules.Supplementary material 1 (PDF 571 kb)Below is the link to the electronic supplementary material.


Reduced E-cadherin expression in RCC promoted cancer progression via WNT/β-catenin signaling pathway activation. E-cadherin/β-catenin provides a valuable prognosis marker for RCC, which may be an effective target for RCC therapy.Reduced expression of E-cadherin was observed in renal cell carcinoma (RCC). However, its potential clinical value and correlation with WNT/β-catenin signaling in RCC progression was still unclear. Immunohistochemical staining was performed in RCC tissue microarray to examine the expression status and prognosis value of E-cadherin and β-catenin. The potential role of E-cadherin in β-catenin translocation was analyzed with immunobloting assays. A significant negative correlation was observed between E-cadherin and β-catenin expression in RCC tissues. E-cadherin inhibits β-catenin translocation from membrane to cytoplasm in RCC tissues, which was an important step for WNT/β-catenin signaling. Reduced E-cadherin expression was associated with poor prognosis. More importantly, E-cadherin-/β-catenin Renal cell carcinoma (RCC) is one of the most common malignant tumor in urinary system with high mortality . IncreasAccumulated evidences supported that the deregulation of cell-to-cell adhesion molecules were involved in RCC progression, which promoted tumor cell invasion and metastatic activity , 5. As a2+-dependent, cell-to-cell adhesion maintains of epithelial cells [As an important member of cadherin complex, β-catenin is involved in Caal cells . The disal cells –13. Moreal cells , 15. Theal cells , 16. How2 = 48. 923, P < 0.001, Figure IHC staining for E-cadherin was performed on TMA slides of RCCs. E-cadherin expression status was classified by the Allred score, score ≥ 4 was positive and score < 4 was negative. Stained E-cadherin protein was located on the cell membrane, which was arranged as brownish yellow granular or linear Figure . E-cadhep < 0.001). Significant lower percentage of E-cadherin expression was observed in clear cell renal carcinoma (7/67) than other types (37/58) (10.45% vs. 63.79%). However, no significant correlation was observed between E-cadherin expression and age, gender, clinical stage, Fuhrman grade and necrosis  compared to other patients , but not for OS . However, no significant correlation was observed with OS  and OS  analyses for disease-free survival (DFS) and overall survival (OS) were performed to investigate the prognostic value of E-cadherin expression in RCC. All the patients involved in our study were followed-up and the median period of 52.27 months (range: 22.8–152.6 months). Disease progression was observed in 21 patients (16.80%) during the period. Distant metastasis was observed with 95.24% of disease-progressed patients (20/21). The survival analysis revealed that E-cadherinTo investigate the underlie mechanism of E-cadherin participated RCC progression, Western blot assays for WNT/β-catenin signaling was performed in four fresh clinical specimens. Increased WNT/β-catenin signaling activation was observed in E-cadherin negative specimens, whereas inhibited WNT/β-catenin signaling in E-cadherin positive ones Figure . FurtherP < 0.01, Figure R2 = 0.120, P < 0.0001, Figure Another IHC staining for β-catenin was performed with TMA specimens of RCC. Positive staining of β-catenin was located in cell membrane/cytoplasm Figure . More imP = 0.030, Table P > 0.05, Table In RCC specimens, significantly increased β-catenin expression was observed in higher Fuhrman grade tissues than lower ones  and OS , but not associated with OS  were incubated respectively at 4°C overnight. Then the sections were stained with peroxidase-conjugated avidin, followed with 3,3-diaminobenzidine tetrahydrochloride (DAB) and hematoxylin blue was used to counterstain. Assigned IHC positive RCC tissues, whose expression were confirmed by Western blot assays, were stained as positive control. Control IgG with equal concentration was used as negative control in each stain.Hematoxylin- and eosin- (H&E) staining of each case was performed to confirm diagnosis and select representative area. TMA was established with representative areas of each tumor with a diameter of 2.0 mm from formalin-fixed, paraffin-embedded tissue blocks. TMA sections were prepared at 5μm of thickness for IHC staining according the following steps: deparaffinized in xylene, hydrated with gradient ethanol, antigen retrieval with 0.01 M sodium citrate for 17 minutes and immersed in 3% HThe evaluation of E-cadherin and β-catenin levels was performed with Allred scoring system . Brown mFresh RCC tissues were collected and homogenized with lysis buffer, which contained 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 0.1% SDS, and 0.1% sodium deoxycholate, pH 7.2. The homogenates were centrifuged at 1000 g for 10 min at 4°C. The membrane and cytosol fractions were prepared with Membrane and Cytosol Protein Extraction Kit according to the manufacturer's instructions . Protein concentration of the supernatant was measured by the Bradford method. Equal amounts of proteins (20 μg) of each sample were separated on 9% SDS-PAGE and electroblotted on polyvinylidene fluoride membranes. Blocked membranes were incubated overnight at 4°C with antibodies against E-cadherin, β-catenin cyclinD1 , Na, K-ATPase  and β-actin  , respectively. The membranes were probed respectively with HRP-conjugated secondary antibodies  at room temperature for 1 h. Immunoreactive protein bands were visualized by use of an enhanced chemiluminescence detection system  according to the manufacturer's protocol.Total cellular mRNA was extracted from fresh RCC tissues using RNAiso Plus Reagent  and reverse-transcribed with 1st Strand cDNA Synthesis Kit  according to the introduction. The RNA concentration was determined by Thermo Scientific NanoDrop 2000 detector. Total of 5 μg mRNA was used for RT-PCR. Primers for E-cadherin, cyclinD1, c-myc and β-actin used in our study were listed as below: CDH1-forward: AAAGGCCCATTTCCTAAAAACCT; Reverse: TGCGTTCTCTATCCAGAGGCT. CCND1-forward: CAA TGACCCCGCACGATTTC; Reverse: CATGGAGGGC GGATTGGAA. MYC-forward: CACACCCACAATTC AGGAAGAG; Reverse: GACGTGCTACAAGGTGGCA. ACTB-forward: TTTTGGCTATACCCTACTGGCA; Reverse: CTGCACAGTCGTCAGCATATC. RT-PCR was performed with a One Step RT-PCR Kit Ver.2 . Each quantity of mRNA expression was corrected by corresponding β-actin mRNA expression.P < 0.05 was considered to be statistically significant.The relationship between E-cadherin and β-catenin expression and clinicopathologic parameters were analyzed using a two- tailed Chi-square test or Fisher's exact test. The correlation between E-cadherin and β-catenin expression status was analyzed with the Spearman's rank test. Survival estimation was performed with the Kaplan-Meier method and log rank test. Univariate or multivariate analysis of prognostic factors was analyzed with Cox proportional hazards regression models. All statistical analyses were performed using the SPSS software system .


Immunohistochemical staining with conventional anti-β-catenin antibody has been applied as a diagnostic tool for desmoid-type fibromatosis (DF). This study aimed to evaluate the diagnostic and prognostic value of immunohistochemical staining with anti-non-phospho β-catenin antibody, which might more accurately reflect the aggressiveness of DF, in comparison to the conventional anti-β-catenin antibody.CTNNB1 mutation status was determined by direct sequencing.Between 2003 and 2015, 40 patients with extra-peritoneal sporadic DF were prospectively treated with meloxicam or celecoxib, a COX-2 inhibitor, therapy. The efficacy of this treatment was evaluated according to Response Evaluation Criteria in Solid Tumors (RECIST). Immunohistochemical staining was performed on formalin-fixed material to evaluate the expression of β-catenin and non-phospho β-catenin, and the positivity was grouped as negative, weak, moderate, and strong. DNA was isolated from frozen tissue or formalin-fixed materials, and the CTNNB1 were detected in 22 (55%) of the 40 cases: T41A (17 cases), S45F (3 cases), and T41I and S45P (1 each). The positive nuclear expression of non-phospho β-catenin showed a significant correlation with positive CTNNB1 mutation status detected by Sanger method (p = 0.025), and poor outcome in COX-2 inhibitor therapy (p = 0.022). In contrast, nuclear expression of β-catenin did not show a significant correlation with either CTNNB1 mutation status (p = 0.43) or outcome of COX-2 inhibitor therapy (p = 0.38).Of the 40 patients receiving COX-2 inhibitor treatment, there was one with complete remission, 12 with partial remission, 7 with stable disease, and 20 with progressive disease. The mutation sites in Nuclear expression of non-phospho β-catenin might more appropriately reflect the biological behavior of DF, and immunohistochemical staining with non-phospho β-catenin could serve as a more useful diagnostic and prognostic tool of COX-2 inhibitor therapy for patients with DF. Desmoid-type fibromatosis (DF), also known as aggressive fibromatosis, is characterized by benign and locally infiltrative fibroblastic tumors. Extra-peritoneal DF are at high risk of local recurrence after planned surgery even with a wide surgical margin range 14.1–68%) [4.1–68% , and surCTNNB1) gene have been reported in 64–85% of sporadic DF. The mutation generally occurs at codon 41 or 45, with T41A , S45F , or S45P (serine to proline) being the most frequent [APC) sporadically. Mutation of APC gene is implicated in the pathogenesis of familial adenomatous polyposis (FAP). Functional impairment of APC complex is induced by these mutations, and β-catenin retains its non-phosphorylated status, translocates and accumulates in the nucleus. In patients with FAP, similar activation of Wnt-β-catenin pathway will occur to that in patients with CTNNB1 mutation. The degree of β-catenin phosphorylation will differ based on the mutation type; codon 41, 45, wild type (WT), or APC gene. Together, the degree of Wnt-β-catenin pathway activation will be dependent on the mutation status of patients with DF. Recent studies have demonstrated that the mutation status of CTNNB1 can help to predict the outcome of surgical [CTNNB1 mutation cannot be evaluated at all institutions. A more appropriate antibody that better reflects desmoid biology is required for pathologists and physicians.In the tumorigenesis of DF, activation of Wnt signaling plays an important role, and aberrant nuclear accumulation of β-catenin has been utilized for pathological diagnosis to differentiate DF from other conditions . Somaticfrequent –18. Thesfrequent . Fewer psurgical , 17 and surgical . In the CTNNB1 mutation status, and help to predict the treatment outcome of patients with DF more precisely than β-catenin.We previously reported that higher nuclear expression of β-catenin correlated with the efficacy of meloxicam treatment for patients with extra-peritoneal sporadic DF . HoweverFifty cases with extra-peritoneal DF were diagnosed in our institutions since 2003. Ten cases were excluded from this study. Five of them did not agree to treatment with COX-2 inhibitor, or had already received “wait and see” follow-up before the referral with status of stable disease. In three, biopsy specimens and formalin-fixed histological preparations were not available for the analyses, while two were excluded due to lack of adequate imaging for evaluation. Finally, this study focused on 40 patients with extra-peritoneal DF, all of whom were prospectively observed with COX-2 inhibitor therapy (meloxicam in 38 and celecoxib in 2). There were no patients with definitive FAP-related DF based on the complete history examinaton. All 40 cases were histologically evaluated and diagnosed with DF by experienced pathologists after thorough discussion. At the beginning of treatment, DF was evaluated with magnetic resonance imaging (MRI) and/or computed tomography (CT) in all cases. Patients were examined physiologically and received MRI and/or CT on an outpatient basis every 3 to 6 months. The efficacy of COX-2 inhibitor treatment was determined based on Response Evaluation Criteria in Solid Tumors (RECIST)  evaluateCTNNB1 was achieved as we previously described [CTNNB1 exon 3: forward 5′–GATTTGATGGAGTTGGACATGG–3′, reverse 5′- TCTTCCTCAGGATTGCCTT -3′, and forward 5′-TGGAACCAGACAGAAAAGCG-3′, reverse 5′- TCAGGATTGCCTTTACCACTC -3′. PCR products were extracted from the isolated bands after gel electrophoresis in 2% agarose, and purified. Direct sequencing was performed with the forward primers, using Applied Biosystems Big Dye Terminator V3.1, and Applied Biosystems 3730× DNA analyzer  at FASMAC Co. Ltd. . Results of the direct sequencing were evaluated using the databases of NCBI-BLAST to determine the mutation sites.Mutation analysis of escribed . BrieflyCTNNB1 mutation status.Tumor specimens were obtained by needle or incisional biopsy in advance of COX-2 inhibitor therapy, fixed in 10% formalin, and embedded in paraffin, and subjected to immunohistochemical study to analyze the expression of β-catenin. According to the previous report , the sliCTNNB1 mutation status. Continuous variables of age and tumor size were compared between the favorable and unfavorable groups, and highly positive and lower positive group for β-catenin and non-phospho β-catenin using the Mann-Whitney U test. All statistical analyses were performed using SPSS statistics 20 . P < 0.05 was considered significant.Fisher exact test and Pearson chi-square test were applied to examine correlations of di- or tri-chotomous variables among the outcome of COX-2 inhibitor therapy, clinical characteristics, results of nuclear staining for β-catenin and non-phospho β-catenin by immunohistochemistry, and Of the 40 patients with extra-peritoneal DF, 16 were male and 24 were female. The mean age was 41.7 years . The location of the tumor was the abdominal wall in 10 patients, other sites in the trunk in 11, extremities in 15, and neck in 4. The diameter of the tumor ranged from 22.5 to 143.7 mm . There were no patients treated with radiotherapy or other conservative treatment including anti-hormonal therapy or low-dose chemotherapy of methotrexate and vinblastine for DF prior to the COX-2 inhibitor therapy.Mean follow up duration from the first visit to the final follow up date of meloxicam therapy was 29.6 months ranging from 2 to 104 months. Of the 40 patients evaluated with RECIST criteria, one patient was classified with CR, 12 with PR, 7 with SD, and 20 with PD. Of the 20 cases with PD, 10 received surgical treatment, and 9 low-dose methotrexate with vinblastine and/or doxorubicin-based chemotherapy, while one refused any other treatment with only continuation of meloxicam therapy.p = 0.089) and tumor size (p = 0.11) had a trend and marginal impact on the prognosis, respectively. Regarding the site of involvement, all 4 cases with neck involvement were assigned to the unfavorable group  and unfavorable (PD) groups, no variable was associated with a significant impact on the prognosis for COX-2 inhibitor therapy, Age (ze p = 0.1 had a tCTNNB1 exon 3. Point mutations existed in 22 of the 40 cases (55%) and were localized to just two codons (41 and 45). The most frequent mutation was replacement of threonine by alanine in codon 41 (T41A), which was detected in 17 cases (43%). Substitution of serine by phenylalanine in codon 45 (S45F) existed in 3 (8%), and that of threonine by isoleucine in codon 41 (T41I) and serine by proline in codon 45 (S45P) in one (3%) each. In the remaining 18 cases (45%), no mutation site was identified in the hot focus (codon 33–45) of CTNNB1 gene (wild type) by Sanger method. Of interest all 4 cases with mutation in codon 45, including 3 cases with S45F mutation, showed progressive disease with COX-2 inhibitor therapy  detected by Sanger sequencing, whereas no correlation was observed between nuclear staining of β-catenin and that of CTNNB1 (p = 0.43). An interesting finding was that fewer cases (n = 1) showed strongly positive for non-phospho β-catenin in WT, T41A, and T41I as compared with that (n = 9) for β-catenin. Among cases with WT and T41A mutated cases, only 30% cases showed strong or moderate positivity for non-phospho β-catenin staining, whereas more than 80% cases showed strong or moderate for β-catenin staining. In contrast, all four cases with mutation of codon 45 (S45F and S45P) showed strong or moderate nuclear positive staining for both β-catenin and non-phospho β-catenin  (Table p = 0.022), whereas that of β-catenin expression did not (p = 0.38) . Nuclear staining intensity of β-catenin and non-phospho β-catenin had no significant correlation with clinical outcome . Analyses of the relationship between staining positivity of non-phospho β-catenin and clinical variables revealed that age (p = 0.013) and tumor size (p = 0.081) had a correlation  Table . Of the 8) Table . On the CTNNB1 gene by ‘priming kinase’ CK1, then at T41, S37, and S33 by GSK3β. Phosphorylation of β-catenin by the APC-axin-GSK-3β complex leads to its degradation by the ubiquitin-proteasome system. With a mutation of any one of these amino acid residues in exon 3 of the CTNNB1 gene or the presence of Wnt stimulation, β-catenin retains non-phosphorylation status, accumulates and translocates into the nucleus, where it binds to the TCF/LEF family of transcription factors and turns on a number of target genes [Wnt signaling plays an important role in the tumorigenesis of DF . In the et genes . In shedP < 0.05) [p = 0.017) to meloxicam therapy based on the 31 cases investigated [p = 0.017, present study, p = 0.38). Lazar et al. observed nuclear β-catenin expression in 98% of specimens, with the percentage of positive nuclear staining cells for β-catenin not correlated with desmoid recurrence, and intensity inversely correlated with the incidence of desmoid recurrence (p < 0.01) [Several studies have reported the correlation between nuclear expression of β-catenin by immunohistochemical examination and the prognosis of patients with DF. Gebert et al. revealed that nuclear β-catenin expression was significantly associated with an increased rate of local recurrence after surgical resection  . They usstigated . Howeverstigated , an incr < 0.01) . The reaCTNNB1 mutation status and clinical outcome of DF has been focused on in several reports. In surgical treatment, DF with the S45F had a greater tendency for local recurrence compared to DF without S45F mutation [Recently, as the treatment algorithm for extra-peritoneal DF has been shifting from primary wide resection to conservative therapy, prognosticators for conservative therapy are also urgently needed. The correlation between mutation , 17, 28.mutation . Consistmutation . These fCTNNB1 mutation have been reported such as age, size and location of the tumor [CTNNB1 mutation, several causes of Wnt signal activation such as APC mutation [Certainly mutation S45F might be a prognosticator for treatment in DF, although the proportion of DF with S45F was only 6–28% of extra-peritoneal DF , 28, 30.he tumor , 34, 35,mutation , activatmutation  and connmutation .CTNNB1 mutation analysis. It is valuable and worth accumulating further studies. Second, non-phospho β-catenin staining could not completely predict the prognosis of patients with COX-2 inhibitor therapy. Nine of the 20 patients with an unfavorable outcome showed negative/weak nuclear non-phospho β-catenin expression, suggesting that Wnt signal activation is not the sole prognosticator in DF. Of clinical characteristics, younger age [CTNNB1 mutation status, and results of immunohistochemical staining. Third, gene mutation was analyzed only in CTNNB1 exon 3 by direct sequencing. Recently Crago et al. performed whole-exome sequence on wild type DF diagnosed with direct sequencing, and reported that 5 of 8 cases with wild type actually had the mutation in Wnt-related gene  [APC mutation despite no history of FAP. Another possible reason is that inadequate fixation time in formalin may cause low quality of extracted DNA, and the false negative results of CTNNB1 mutation status because 22 cases were analyzed using FFPE samples. Further studies with additional molecular analyses will be required to better determine the correlation of prognosis and patient characteristics, mutation status, and immunohistochemical findings.There were several limitations in our study. First, the number of patients was small due to the rarity of DF, and thus it may have been underpowered to detect a significant difference between groups. However, the cohort of this study was prospectively treated with COX-2 inhibitor and subjected to nger age , 35 and nger age , 35 were in APC) . SimilarWe demonstrate for the first time that nuclear non-phospho β-catenin expression could predict the treatment efficacy of with COX-2 inhibitor in patients with sporadic DF. Larger prospective clinical studies and molecular analyses are still necessary to determine the significance of this non-phospho β-catenin staining.


Cancer cells are the site of numerous metabolic and thermodynamic abnormalities. We focus this review on the interactions between the canonical WNT/beta-catenin pathway and peroxisome proliferator-activated receptor gamma (PPAR gamma) in cancers and their implications from an energetic and metabolic point of view. In numerous tissues, PPAR gamma activation induces inhibition of beta-catenin pathway, while the activation of the canonical WNT/beta-catenin pathway inactivates PPAR gamma. In most cancers but not all, PPAR gamma is downregulated while the WNT/beta-catenin pathway is upregulated. In cancer cells, upregulation of the WNT/beta-catenin signaling induces dramatic changes in key metabolic enzymes that modify their thermodynamic behavior. This leads to activation of pyruvate dehydrogenase kinase1 (PDK-1) and monocarboxylate lactate transporter. Consequently, phosphorylation of PDK-1 inhibits the pyruvate dehydrogenase complex (PDH). Thus, a large part of pyruvate cannot be converted into acetyl-coenzyme A (acetyl-CoA) in mitochondria and only a part of acetyl-CoA can enter the tricarboxylic acid cycle. This leads to aerobic glycolysis in spite of the availability of oxygen. This phenomenon is referred to as the Warburg effect. Cytoplasmic pyruvate is converted into lactate. The WNT/beta-catenin pathway induces the transcription of genes involved in cell proliferation, i.e., MYC and CYCLIN D1. This ultimately promotes the nucleotide, protein and lipid synthesis necessary for cell growth and multiplication. In cancer, activation of the PI3K-AKT pathway induces an increase of the aerobic glycolysis. Moreover, prostaglandin E2 by activating the canonical WNT pathway plays also a role in cancer. In addition in many cancer cells, PPAR gamma is downregulated. Moreover, PPAR gamma contributes to regulate some key circadian genes. In cancers, abnormalities in the regulation of circadian rhythms (CRs) are observed. CRs are dissipative structures which play a key-role in far-from-equilibrium thermodynamics. In cancers, metabolism, thermodynamics and CRs are intimately interrelated. Schrödinger in his famous book “What is life”  provided. The canonical WNT activity is reflected by elevated levels of beta-catenin in the nucleus and/or cytoplasm, which can be detected by means of immunohistochemical staining, Western blotting and semiquantitative RT-PCR [The canonical WNT/beta-catenin pathway plays an important role in metabolism, embryonic development, cell fate, and epithelial-mesenchymal transition (EMT) . The cae RT-PCR . Its dyse RT-PCR –31. The e RT-PCR –35 , since the pathway via free fatty acid beta-oxidation is less efficient (ATP/O2 = 5.6). This takes about 11% more O2 to produce the same amount of ATP from fatty acids as it does from glucose. Moreover, PDK-1 and 2 enhance angiogenesis [In cancer, the behavior of two key enzymes involved in glucose metabolism is modified leading to the Warburg effect. Activation of PDK-1 is required for the Warburg aerobic glycolysis. Upregulation of WNT/beta-catenin signaling activates both PDK-1 and MCT-1 , 58. PDKogenesis , 67. Bloogenesis . Conversogenesis . PDKs alogenesis  and are ogenesis . SeveralIn colon cancer, activation of WNT/beta-catenin signaling decreases the oxidative metabolism in the TCA cycle and promotes cell proliferation . In addiPhosphofructokinase (PFK), an allosteric enzyme, is responsible for glycolytic oscillations. PFK can lead to instabilities beyond which a new state can be organized in time and in space . A positCancer cells are characterized by increased glucose consumption. High serum glucose levels may modulate cancer-related processes. Glucose itself can directly impact the canonical WNT pathway . High glIt has been recently described a novel antitumoral mechanism of vitamin C . MutatioFrom a thermodynamic viewpoint, the lawless-disorderly cancer growth and the orderly fetal growth share some similar features . HypoxicCTNNB1 which encodes beta-catenin, have been reported in numerous cancers such as breast colorectal, melanoma, prostate and lung tumors. WNT 1, WNT2 and WNT7A ligand-proteins are overexpressed in glioblastoma, esophageal cancer and ovarian cancer respectively. Proteins of the TCF/LEF family and WNT5A may also induce cancer. Repression of WNT/beta-catenin signaling can prevent EMT and inhibit metastasis. Mutations of the WNT pathway components are associated with many cancers, particularly with colorectal cancer. APC deficiency and beta-catenin mutations upregulate the WNT/beta-catenin pathway and prevent beta-catenin degradation. This leads to excessive stem cell renewal and cell proliferation that predisposes to tumor genesis particularly for colorectal cancer [APC gene mutations favors cell proliferation [WNT/beta-catenin signaling has been found to be activated in cancers , 93. WNTl cancer . Nuclearl cancer , and actferation . Mutatioferation .Several studies have presented evidence for a protective role of PPAR gamma against cancer. In colon cancer, PPAR gamma downregulates the oncogene beta-catenin and suppresses cell proliferation . In contHyperactivation of phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) pathway is associated with an increased rate of glucose metabolism in tumor cells . AKT sigThe phosphatase and tensin homologue (PTEN) represents the main brake of the PI 3′-OH kinase (PIK3)-AKT pathway . PI3K geCtnnb1, Ptgs2, Ccnd1, Mmp9) are significantly upregulated after PGE2 use. PPAR gamma and proinflammatory enzyme pathways are interrelated. Decreased expression of PPAR gamma and high levels of COX-2 have been reported in many cancers [Several studies have established the role of prostaglandin E2 (PGE2) by activating the WNT/beta-catenin pathway. The link between PGE2 and the canonical WNT pathway suggests that chronic inflammation induced by a prolonged increase of PGE2 could lead to activation of WNT signaling resulting in cell proliferation and cancer. PGE2 enhances the beta-catenin-dependent transcription , 143. PG cancers . TZDs de cancers .CRs can be defined as endogenous, entrainable free-running periods that last approximately 24 h. CRs are far-from-equilibrium dissipative structures and are due to a negative feedback produced by a protein on the expression of its own gene –152. TheBMAL1 function modifies the behavior of genes involved in the canonical WNT pathway [Circadian rhythms govern numerous physiological and metabolic functions . Thus, C pathway . Beta-ca pathway . A decea pathway , prostat pathway , pancrea pathway , colorec pathway , chronic pathway , and gli pathway , 169.PPAR gamma in mouse impairs diurnal rhythms [BMAL1 and Rev-erb alpha. Mice lacking PGC-1 alpha show changes in CRs and metabolism [Peroxisome proliferator-activated receptors interferes with the mammalian clock and energy metabolism . PPARs a rhythms . PPAR gatabolism . PGC-1 atabolism . PGC-1 atabolism . PPAR alPDK and MCT-1 are also target genes of beta-catenin, explaining the significant decrease in the transformation of pyruvate into acetyl-CoA in mitochondria and the formation of intracellular lactate, which will be extruded out of the cell. This is referred to as aerobic glycolysis or the Warburg phenomenon. The expression of PPAR gamma is decreased due to the overactivation of WNT/beta-catenin signaling. Circadian rhythms, dissipative structures which are governed by the laws of far-from-equilibrium thermodynanics are disrupted in cancers. They are influenced by both the WNT/beta-catenin pathway and PPAR gamma. Changes in thermodynamics, metabolism and circadian rhythms are tightly linked in cancers.Cancers exhibit thermodynamic and metabolic alterations and abnormal CRs. In many cancers but not all, the canonical WNT/beta-catenin pathway is upregulated, while PPAR gamma is downregulated, the two systems behaving in an opposite manner. Overactivation of the WNT pathway results in cell proliferation due to the activation of certain target genes of beta-catenin, such as MYC and CYCLIN D1. This promotes protein synthesis and angiogenesis.


Maternally expressed gene 3 (MEG3) encodes an lncRNA which is suggested to function as a tumor suppressor and has been showed to involve in a variety of cancers. Herein, our findings demonstrate that MEG3 inhibits the malignant progression of liver cancer cells in vitro and in vivo. Mechanistically, MEG3 promotes the expression and maturition of miR122 which targets PKM2. Therefore, MEG3 decreases the expression and nuclear location of PKM2 dependent on miR122. Furthermore, MEG3 also inhibits CyclinD1 and C-Myc via PKM2 in liver cancer cells. On the other hand, MEG3 promotes β-catenin degradation through ubiquitin–proteasome system dependent on PTEN. Strikingly, MEG3 inhibits β-catenin activity through PKM2 reduction and PTEN increase. Significantly, we also found that excessive β-catenin abrogated the effect of MEG3 in liver cancer. In conclusion, our study for the first time demonstrates that MEG3 acts as a tumor suppressor by negatively regulating the activity of the PKM2 and β-catenin signaling pathway in hepatocarcinogenesis and could provide potential therapeutic targets for the treatment of liver cancer. Notably, genetic variants and imprint change in MEG3 may contribute to the development and risk of cancer7. Moreover, MEG3 increases autophagy8, and epigenetic repression of MEG3 represses the p53 pathway and enhances Wnt/β-catenin signaling10. In addition, MEG3 produces an antitumor effect in several cancers12. Furthermore, MEG3 functions as a competing endogenous RNA to regulate cancer progression13 and TGF-β pathway genes through the formation of RNA–DNA triplex structures14. Strikingly, excessive MEG3 promotes osteogenic differentiation of mesenchymal stem cells from multiple myeloma patients by targeting BMP4 transcription15.Recent research has found that long noncoding RNAs (lncRNAs) were involved in various human cancers. Maternally expressed gene 3 (MEG3) has been shown to be involved in a variety of cancers and is downregulated in most cancers and affects cell proliferation, progression, and prognosis19. In particular, miR-122 reverses the drug resistance and hepatotoxicity in hepatocellular carcinoma cells through regulating the tumor metabolism21. Pyruvate kinase muscle isozyme M2 (PKM2) is a limiting glycolytic enzyme that catalyzes the final step in glycolysis, which is key in tumor metabolism and growth23. Moreover, PKM2 plays a pivotal role in the growth, survival, and metabolic reprogramming of cancer cells25. Notably, loss of SIRT2 function in cancer cells reprograms their glycolytic metabolism via PKM2 regulation26. In addition, our previous study indicates that double mutant P53 (N340Q/L344R) promotes hepatocarcinogenesis mediated by PKM227. Phosphatase and tensin homolog (PTEN) is one of the powerful switches for the conversion between tumor suppressors and oncogenes. A number of studies have suggested that PTEN may alter various functions of certain oncogenic proteins33. Strikingly, PTEN opposes malignant transformation of pre-B cells and breast cells35. In particular, the PI3K-PTEN-AKT-mTOR pathway is a central controller of cell growth and a key driver for human cancer36. β-catenin (encoded by CTNNB1) is a subunit of the cell surface cadherin protein complex that acts as an intracellular signal transducer in the WNT signaling pathway. Many hepatic tumors such as hepatocellular adenomas, hepatocellular cancers, and hepatoblastomas have mutations in β-catenin that result in constitutive activation of β-catenin37. Also, Wnt/β-catenin/TCF-4 signaling is crucial for the proliferation and self-renewal maintenance of cancer stem cells41. Strikingly, MSK1-mediated β-catenin phosphorylation confers resistance to PI3K/mTOR inhibitors in glioblastoma42.miR-122 is involved in human cancer proliferation, invasion, and progressionIn the present study, we indicate that MEG3 inhibits the malignant progression of liver cancer cells in vitro and in vivo. Our study for the first time demonstrated that MEG3 acts as a tumor suppressor by negatively regulating the activity of the PKM2 and β-catenin pathway in hepatocarcinogenesis and may provide potential therapeutic targets for the treatment of liver cancer.2 incubator at 37 °C. Plasmids pGFP-V-RS, pCMV6-A-GFP, pCMV6-XL5-β-catenin, pCMV6-XL5-PTEN, pGFP-V-RS-PTEN, pGFP-V-RS-β-catenin, and pMiR-Target were purchased from Origene . pEGP-miR122(BioLab), pCMV6-A-GFP–MEG3 was constructed in our lab.Human liver cancer line Hep3B was maintained in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco) in a humidified atmosphere of 5% COR 2000 (Invitrogen) according to manufacturer’s instructions. For screening stable cell lines, 48 h after transfection, the cells were plated in the selective medium containing G418  or Puromycin  for about 4 weeks or so, and the GFP-positive cells were selected and the selective media were replaced every 3 days.Cells were transfected with DNA plasmids using transfast transfection reagent lipofectamine17-18, random primers, and a SuperScript First-Strand Synthesis System (Invitrogen). The PCR reaction was performed in 36 cycles with each cycle consisting of a denaturation step , an annealing step (56 °C (MEG3) or 55 °C (PKM2 and β-actin) or 58 °C (pre-miR122) for 30 s), and an elongation step . PCR primers: MEG3:P1: 5′-TCCATGCTGAGCTGCTGCCAAG-3′; P2: 5′- AGTCGACAAAGACTGACACCC-3′; PKM2P1: 5′-GCCACCATGTCGAAGCCCCATA-3′; P2: 5′-TCACGGCACAGGAACAACACGC-3′; pre-miR122: P1: 5′-TTGCCTAGCAGTAGCTATTT-3′; P2: 5′GGCTACAGAGTTTCCTTAGC-3′. β-actin was used as an internal control.Total RNA was purified using Trizol (Invitrogen) according to manufacturer’s instructions. cDNA was prepared by using oligonucleotide (dT)Ct values for miR122 were calculated and normalized to Ct values for U6 snRNA.Total RNA was isolated from cultured cells using Trizol  according to the manufacturer’s protocol. Real-time RT-PCR-based detection of mature miR-122 and U6 snRNA was achieved with the miRNA Detection kit  and miR122 specific upstream primers . qRT-PCR was performed with a StepOne Plus real-time PCR system (Applied Biosystems). The real-time PCR reaction was performed in 40 cycles with each cycle consisting of a denaturation step  and an annealing step (60 °C for 30 s). Each sample was run in triplicate. g for 20 min at 4 °C after sonication on ice, and the supernatant was separated. After being boiled for 5–10 min in the presence of 2-mercaptoethanol, the samples containing cellulars proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane, blocked in 10% dry milk-TBST (20 mM Tris-HCl (pH 7.6), 127 mM NaCl, 0.1% Tween 20) for 1 h at 37 °C. Following three washes in Tris-HCl pH 7.5 with 0.1% Tween 20, the blots were incubated with 0.2 µg/ml of antibody (appropriate dilution) overnight at 4 °C. Following three washes, the membranes were then incubated with secondary antibody for 60 min at 37 °C or 4 °C overnight in TBST. Signals were visualized by ODYSSEY infrared imaging system . IRDye 680LT/IRDye 800CW secondary antibodies  were purchased from LI-COR Scientific Company. Standard Western blotting procedures were used with the following antibodies: anti-PCNA , anti-CTCF , anti-CREB , anti-RNA polII(Abcam), anti-H3K9me3 (Abcam), anti-H3K36me3 (Abcam), anti-PKM2 (Abcam), anti-ERK1/2 (Abcam), anti-pPKM2(ser37) , anti-Histone H3 (Abcam), anti-H3K9Ac (Abcam), anti-METTL3 , anti-AKT (Abcam), anti-pAKT (Abcam), anti-GSK3β(ser9), anti-GSK3β(Tyr216), anti-GSK3β , anti-β-catenin , anti-HA, anti-Skp2 , anti-TCF4 , anti-LEF , anti-PTEN , anti-LEF , anti-TCF4 , anti-C-myc , and anti-cyclinD1 .The logarithmically growing cells were washed twice with ice-cold phosphate-buffered saline  and lysed in a RIPA lysis buffer. Cells lysates were centrifuged at 12,000Cells were lysed in 1 ml of whole-cell extract buffer A ; 500 μl cell lysates were used for IP with antibody. In brief, protein was pre-cleared with 30 μl protein G/A-plus agarose beads  for 1 h at 4 °C and the supernatant was obtained after centrifugation (5000 rpm) at 4 °C. Pre-cleared homogenates (supernatant) were incubated with 2 µg of antibody and/or normal mouse/rabbit IgG with rotation for 4 h at 4 °C. The immunoprecipitates were incubated with 30 μl protein G/A-plus agarose beads by rotation overnight at 4 °C, and then centrifuged at 5000 rpm for 5 min at 4 °C. The precipitates were washed five times for 10 min with beads wash solution , resuspended in 60 µl 2 × SDS-PAGE sample loading buffer, and incubated for 5–10 min at 100 °C. Western blotting was performed with related antibodies.2, 10 mM HEPES [pH 7.0], 0.5% NP40, 1 mM DTT, 100 units/ml RNase OUT (Invitrogen), 400 μM vanadyl–ribonucleoside complex, and protease inhibitors (Roche), clarified and stored at −80 °C. Ribonucleoprotein particle-enriched lysates were incubated with protein A/G-plus agarose beads  together with the antibody or normal mouse or rabbit IgG for 4 h at 4 °C. Beads were subsequently washed four times with 50 mM Tris-HCl (pH 7.0), 150 mM NaCl, 1 mM MgCl2, and 0.05% NP-40, and twice after addition of 1 M urea. Immunoprecipitates were digested with proteinase K  and mRNAs were isolated and purified, and then RT-PCR was performed.Cells were lysed  in 100 mM KCl, 5 mM MgCl2, 3 mM DTT, and 5% glycerol). Twenty-five pmol unlabeled cold DNA motifs (a 250-fold excess) were added in the competition assays. Reactions were carried out for 30 min incubation at room temperature, followed by overnight incubation at 4 °C. Reaction mixtures were loaded onto 6% TBE polyacrylamide gels and separated in 0.5%×TBE at 100 v on ice until the dye front migrated two-thirds of the way to NC membranes and Western blotting was performed for anti-biotin.Cells were washed and scraped in ice-cold PBS to prepare nuclei for electrophoretic gel mobility shift assay with the use of the gel shift assay system modified according to the manufacturer’s instructions (Promega). In brief, consensus oligonucleotides for damage or repair DNA were biotin-labeled (hot probe). Each binding reaction was carried out with 1 µg biotinylated dsDNA probe and 200 µg purified nuclear protein in 20 µl of binding buffer containing 0.5 mg/ml poly(dI:dC) , and luciferase activities of cell extracts were measured with the use of the Dual luciferase assay system (Promega) according to manufacturer’s instructions. Luciferase activity was measured and normalized for transfection efficiency with Renilla luciferase activity.Cells were cross-linked with 1% (v/v) formaldehyde for 10 min at room temperature and stopped with 125 mM glycine for 5 min. Cross-linked cells were washed with PBS, resuspended in lysis buffer, and sonicated for 8–10 min in a SONICS. Chromatin extracts were pre-cleared with Protein-A/G-Sepharose beads, and immunoprecipitated with specific antibody on Protein-A/G-Sepharose beads. After washing, elution, and de-cross-linking, the ChIP DNA was detected by PCR. The following primer pairs were used: C-myc CHIP promoter: P1: 5′-TAACTCGCTGTAGTAATTCC-3′; P2: 5′-CCCTATGGGCAAAGTTTCGT-3′; CyclinD1 CHIP promoter: P1: 5′-GAAGAGTCTCCAGGCTAGAA-3′; P2: 5′-TTGTAGCCTGGAGACTCTTC-3′.3 were seeded into 96-well culture plates in 100 μl culture medium containing 10% heat-inactivated fetal calf serum (FCS). Before detection, 10 μg/well cell proliferation reagent CCK8 was added and incubated for 4 h at 37 °C and 5% CO2. Cell growth curve was based on the corresponding normalized values of OD450 and each point represents the mean of three independent samples.Cells were synchronized in G0 phase by serum deprivation and then released from growth arrest by re-exposure to serum, and then cells were grown in complete medium for assay. The cell proliferation reagent CCK8 was purchased from Roch and the operation was carried out according to the manufacturer's instruction. In brief, cells at a concentration 4 × 102 cells were plated on a 10-cm dish, then 10 ml DMEM containing 10% FBS was added into each 10-cm dish of the three replicates. Then these dishes were incubated at 37 °C in a humidified incubator for 10 days. The cell colonies on the dishes were stained with 1 ml of 0.5% Crystal Violet for more than 1 h and the colonies were counted.5 × 106 cells in 100 μl of PBS. The mice were observed over 4 weeks, and then sacrificed to recover the tumors. The wet weight of each tumor was determined for each mouse. A portion of each tumor was fixed in 4% paraformaldehyde and embedded in paraffin for histological hematoxylin–eosin (HE) staining.Four-weeks-old male athymic Balb/C mice were purchased from Shi Laike Company  and maintained in the Tongji animal facilities approved by the China Association for Accreditation of Laboratory Animal Care. The athymic Balb/C mice were injected in the armpit area subcutaneously with Hep3B suspension of 1 × 10All methods were carried out in “accordance” with the approved guidelines. All experimental protocols “were approved by” a Tongji university institutional committee. Informed consent was obtained from all subjects. The use of mice  was reviewed and approved by the China national institutional animal care and use committee”.P < 0.01). We further performed plate colony formation assay and observed a significant decrease in colony formation efficiency rate in excessive MEG3  ; when MEG3 was overexpressed, the xenograft tumor size decreased approximately one-fifth compared to the corresponding control group . Moreover, compared to control, xenograft tumors contained less of poorly differentiated cells in MEG3 overexpression group , pCMV6-A-GFP-MEG3 (MEG3), respectively. As shown in Fig. 01) Fig. . To exploup Fig. , upper. To address whether MEG3 influences the expression and maturation of miR122, we performed related experiments in MEG3 or miR122 overexpressing Hep3B cell line. As shown in Fig. P < 0.01)  Fig. . Further01) Fig. . Thus, t01) Fig. . Signifi01) Fig. . However01) Fig. . Moreove01) Fig.  and the 01) Fig. . Therefo01) Fig. . Further01) Fig. . Strikin01) Fig. . Thus, M01) Fig. . Further01) Fig. . Ultimat01) Fig. . Taken tP < 0.01)  Fig. , however01) Fig. . The exp01) Fig. . Therefo01) Fig. . Interes01) Fig. . Thus, e01) Fig. . Further01) Fig. . Strikin01) Fig. . Ultimat01) Fig. . Excessi01) Fig. . Taken tTo explore whether MEG3 influenced β-catenin activity, we analyzed the activity of β-catenin co-activators LEF and TCF-4 in liver cancer cells. As shown in Fig. P < 0.01). However, HULC plus β-catenin did not significantly alter the growth of liver cancer cells (P > 0.05). Moreover, MEG3 overexpression significantly decreased the BrdU positive rate compared to the control cells . However, MEG3 plus β-catenin did not significantly alter the BrdU positive rate of liver cancer cells  . However, MEG3 plus β-catenin did not significantly alter the colony formation rate of liver cancer cells  . However, MEG3 plus β-catenin did not significantly alter the xenograft tumor weight . On the other hand, when MEG3 was overexpressed, the average xenograft tumor appearance time was significantly increased compared to the corresponding control group . However, MEG3 plus β-catenin did not significantly alter the xenograft tumor appearance time  . However, MEG3 plus β-catenin did not significantly alter the PCNA positive rate of liver cancer cells  . As shown in Fig. 05) Fig. . We furt05) Fig. . Further05) Fig. . Moreove05) Fig. . Compare05) Fig. . This suIt has been confirmed that MEG3 encodes an lncRNA which is suggested to function as a tumor suppressor and has been shown to involve in a variety of cancers. Our studies are now indicated to evaluate the effects of MEG3 in liver cancer cells. Our findings demonstrate that MEG3 inhibits the malignant progression of liver cancer cells in vitro and in vivo. Mechanistically, MEG3 promotes the expression and maturation of miR122 which targets PKM2. Therefore, MEG3 decreased the PKM2 expression and nuclear location dependent on miR122. Furthermore, MEG3 inhibited CyclinD1 and C-Myc via PKM2 in liver cancer cells. Strikingly, MEG3 promotes β-catenin degradation through ubiquitin–proteasome system dependent on PTEN. Moreover, MEG3 inhibits β-catenin activity through PKM2 reduction and PTEN increase. Furthermore, we found that excessive β-catenin rescued the effect of MEG3 in liver cancer Fig. . To our 43. Crosstalk between MEG3 and miR-1297 regulates the growth of testicular germ cell tumor through PTEN/PI3K/AKT pathway44. MEG3 may be an underlying therapeutic target for LUAD functioning as ceRNAs for the regulation of miRNA-mRNA in lung adenocarcinoma45. In addition, MEG3 was decreased in primary endometrial stromal cells (ESCs) in response to TGF-β1 stimulation46.To date, accumulating evidence indicates that MEG3 plays a critical role in cancer progression and metastasis. Previous studies suggested that MEG3 functioned through the activation of p53; however, the functional properties of MEG3 remain obscure and their relevance to human diseases is under continuous investigation1 MEG3 is downregulated and is postively associated with miR122, PTEN and negatively associated with PKM2, β-catenin expression in human liver cancer tissue2. MEG3 inhibits malignant progression of liver cancer cells in vitro and in vivo. Our observations demonstrated that MEG3 is crucial for the inhibition of cell growth and viability in liver cancer cells. According to the aforementioned findings, MEG3 is a tumor suppressor.Evidently, our results indicate that the involvement of MEG3 inhibition of liver cancer cell growth is supported by results from two parallel sets of experiments:47. Furthermore, miR122 is implicated as a regulator of physiological and pathophysiological processes in the liver. Gα12 overexpressed in hepatocellular carcinoma reduces microRNA-122 expression via HNF4α inactivation, which causes c-Met induction48. A quantitative mathematical model of HCV-induced miR-122 sequestration proposes that such miR122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV49.Of significance, our findings clearly showed that MEG3 promotes the expression and maturation of miR122 which targets PKM2 and inhibits the expression of PKM2. Studies indicate that alcoholic hepatitis accelerates early hepatobiliary cancer by increasing stemness and miR122-mediated HIF-1α activation1: MEG3 decreased the PKM2 expression dependent on miR1222. MEG3 inhibits nuclear localization and function of PKM2 dependent on miR1223. MEG3 inhibits the expression of cyclinD1 and C-Myc via PKM2. These findings are noteworthy, given that PKM2 is and functions as a key oncogene to mediate various biological processes including cell proliferation and differentiation. Moreover, PKM2 is associated with cancer differention51. Pyruvate kinase M2 activates mTORC1 by phosphorylating AKT1S152 and PKM2 promotes tumor angiogenesis by regulating HIF-1α through NF-κB activation53. In particular, cytosolic PKM2 stabilizes mutant EGFR protein expression through regulating HSP90-EGFR association54. PKM2 promotes stemness of breast cancer cell by through Wnt/β-catenin pathway55. In addition, miR675 upregulates lncRNA H19 through activation of EGR1 in human liver cancer56. However, a study failed to observe PKM2-dependent transfer of phosphate from ATP directly to protein57. Furthermore, our findings indicated that MEG3 inhibited the expression of C-myc, whereas C-myc decides the reprogramming metabolism of cancer58.Accordingly, reduction in PKM2 may partly contribute to MEG3-mediated inhibition of liver cancer cell growth. Our findings in this study provide a novel evidence for an active role of PKM2 in MEG3-mediated inhibition of liver cancer cell growth. This assertion is based on several observations1: MEG3 increased the expression and phosphorylation of PTEN2. MEG3 promotes β-catenin degradation through ubiquitin–proteasome system dependent on PTEN3. MEG3 inhibits β-catenin activity through PKM2 reduction and PTEN increase in liver cancer cells4. MEG3 inhibited cell growth, colony formation ability, and cell growth in vivo. However, β-catenin overexpression abrogated the MEG3 action. β-catenin determines MEG3 suppressor function in liver cancer cells.Strikingly, we also demonstrated that MEG3 is closely associated with PTEN and β-catenin in liver cancer cells. This assertion is based on several observations60. Notch promotes tumor metastasis in a prostate-specific PTEN-null mouse model61. miR-18a promotes cell proliferation of esophageal squamous cell carcinoma cells by increasing cylin D1 via regulating PTEN-PI3K-AKT-mTOR signaling axis62. Furthermore, PTEN is a key molecular controller of the PI3K signaling, and PI3K-PTEN dysregulation leads to mTOR-driven upregulation of the core clock gene BMAL1 in normal and malignant epithelial cells63. In particular, PTEN negatively regulates mTORC2 formation and signaling in grade IV glioma via Rictor hyperphosphorylation at Thr1135 and directs the mode of action of an mTORC1/2 inhibitor64. In addition, SOX7 co-regulates Wnt/β-catenin signaling with Axin-265 and FAK promotes osteoblast progenitor cell proliferation and differentiation by enhancing Wnt signaling66. Moreover, FOXKs promote Wnt/β-catenin signaling by translocating DVL into the nucleus67 and ECM1 regulates tumor metastasis and CSC-like property through stabilization of β-catenin68. A report shows that microRNA-153 promotes β-catenin activation in hepatocellular carcinoma through the suppression of WWOX69. Also, there is a regulation function between Wnt/β-catenin signaling and PI3K/Akt survival pathway70.It is well known that PTEN is a lipid phosphatase that converts phosphatidylinositol 3,4,5-phosphate (PIP3) to phosphatidylinositol 4,5-phosphate (PIP2) and plays a critical role in the regulation of tumor growthThe present study depicts a novel evidence for MEG3 that plays inhibiting tumorigenesis roles by downregulating PKM2 and β-catenin in liver cancer cells, which may have potential therapeutic significance. Alteration of the expression of lncRNAs MEG3 may also mediate changes at an epigenetic level to affect gene expression and contribute to inhibiting hepatocarcinogenesis. MEG3 overexpression in combination with blocking PKM2 and β-catenin might represent a promising treatment strategy targeting tumors. Our study for the first time demonstrated that MEG3 acts as a tumor suppressor by negatively regulating the activity of PKM2 and β-catenin in hepatocarcinogenesis and might serve as a prognostic biomarker and molecular therapeutic target.


Induced pluripotent stem cells (iPSCs) have provided new opportunities for motor neuron disease (MND) modeling, drug screening, and cellular therapeutic development. Among the various types of iPSCs, urine-derived iPSCs have become a promising source of stem cells because they can be safely and noninvasively isolated and easily reprogrammed. Here, for the first time, we differentiated urine-derived iPSCs (urine-iPSCs) into motor neurons (MNs) and compared the capacity of urine-iPSCs and cord-blood-derived iPSCs (B-iPSCs) to differentiate into MNs. With the use of small molecules, mature MNs were generated from urine-iPSCs as early as 26 days in culture. Furthermore, in coculture with muscle cells, MNs projected long axons and formed neuromuscular junctions (NMJs). Immunofluorescence and PCR confirmed the expression levels of both MN and NMJ markers. The comparison of the ratios of positive labeling for MN markers between urine-iPSCs and B-iPSCs demonstrated that the differentiation potentials of these cells were not significantly different. The abovementioned results indicate that urine-iPSCs are a new, promising source of stem cells for MND modeling and further cellular therapeutic development. Motor neuron diseases (MNDs) selectively affect motor neurons (MNs), which project axons to muscles and control voluntary actions. Patients with MNDs may present a range of symptoms, such as muscular weakness, atrophy, and hyperreflexia, which ultimately lead to death . No effeThe human iPSC lines used in this study included two urine cell-derived iPSC lines, UE017 and UC005, obtained from the Chinese Academy of Sciences, Guangzhou Institute of Biomedicine and Health . As a coμM EDTA (Invitrogen) for 5 min and then passaged in Matrigel-coated plates in 1 : 6. The following day, the stem cell medium mTeSR was replaced with neural differentiation medium (NDM) with the addition of 3 μM CHIR99021 , 2 μM DMH1 (Sigma), and 2 μM SB341542 . NDM includes Dulbecco's Modified Eagle Medium (DMEM/F12), Neurobasal Medium at a 1 : 1 concentration, 0.5 × N2, 0.5 × B27, 0.1 mM ascorbic acid (Sigma), 1 × Glutamax, and 1 × antibiotic-antimycotic . The medium was changed every other day. IPSCs maintained under these conditions for 6 days differentiated into neuroepithelial progenitors (NEPs). On day 7, the NEPs were treated with dispase (1 mg/ml) for 5 min and then gently resuspended with NDM, including 1 μM CHIR, 2 μM DMH1, 2 μM SB, 0.1 μM retinoic acid , and 0.5 μM purmorphamine , and were plated on Matrigel-coated plates at a 1 : 3 concentration. The culture medium was changed every other day for 6 days, and on day 12, the NEPs differentiated into motor neuron progenitors (MNPs), which aggregated in rosettes. To induce MNPs into MNs, the rosettes were lifted with dispase (1 mg/ml) and then cultured in suspension in NDM with 0.5 μM RA, 0.1 μM Pur, 10 ng/ml brain-derived neurotrophic factor , 10 ng/ml glial cell line-derived neurotrophic factor , and 10 ng/ml insulin-like growth factor  for 6 days. On day 19, HB9-positive MN spheres formed. To induce further maturation, these MN spheres were dissociated with accutase (Invitrogen) into single neurons and cultured adherently with NDM, including 0.5 μM RA, 0.1 μM Pur, 0.1 μM Compound E , and the abovementioned neurotrophic factors, for more than 7 days. Then, MNs differentiated into mature MNs.Du's protocol for the differentiation of iPSCs into MNs was used with slight modification  7]. For. For7]. C2C12 cells, a mouse myoblast cell line, were obtained from the China Center for Type Culture Collection and cultured in growth medium ) until the cells reached 60–70% confluence. Then, the medium was changed to differentiation medium (DMEM/F12 with 2% horse serum (HS)), and myoblasts cultured in this medium for 4 days differentiated into skeletal muscle cells. On day 19, UC005-derived MN spheres were dissociated into single MNs, the MNs were added to each dish of muscle cells, and the medium was replaced with NDM, which was supplemented with RA, Pur, BDNF, GDNF, and IGF. Within 1-2 days, the MNs projected axons, and after more than 1 week in culture, the MNs formed NMJs with the muscle cells.α-BTX  for 1 hour at 37°C before fixation. The cells were then washed 2 times in PBS and fixed with 4% PFA. All cell samples were observed using an Olympus fluorescence microscope.The cultured cells were placed on 12 mm cover slips, fixed in 4% paraformaldehyde  for 10 min at room temperature, washed three times with phosphate-buffered saline , and treated with a permeabilizing and blocking buffer  for 1 hour at room temperature. Then, the cells were incubated with the following primary antibodies: OCT4 , Nanog , SSEA4 , TRA-1-60 , SOX1 , SOX2 , Nestin , Olig2 , Pax6 , HB9 , Islet1 , ChAT , and TuJ1 . All antibodies were diluted in antibody dilution buffer , and the cells were incubated with the antibodies overnight at 4°C. After three washing steps, the cells were incubated for 1 hour at room temperature with secondary antibodies: Alexa Fluor (488 or 555) donkey anti-mouse, donkey anti-rabbit, and donkey anti-goat. For the detection of AChR, the cocultured cells were incubated with Alexa Fluor 555-conjugated μg isolated RNA was reverse-transcribed to cDNA using the PrimeScript™ RT Master Mix . The PCRs contained 1.5 μl cDNA, 10 μl Ex Taq (TAKARA), 0.8 μl primers . TRIzol (Sigma), chloroform, isopropanol, and DNase I were used for total RNA extraction. Then, 0.8  primers , and 7.7t-test, and the results are presented as the means ± standard errors of the mean. The P values ∗P < 0.01, ∗∗P < 0.001, and ∗∗∗P < 0.0001 were considered significant.GraphPad Prism 7 Software (GraphPad Software) was used for statistical analysis. The presented results are from three independent experiments. Statistical significance was determined with the Before initiation of the differentiation process, we first analyzed the pluripotent properties and purity of the iPSCs. Both urine-iPSCs and B-iPSCs exhibited uniform undifferentiated morphology, including a round shape, large nucleoli, scant cytoplasm, and organized colonies, similar to the features of embryonic stem cells (ESCs)  8]. All. All8]. β) signaling pathways . Th. Thβ) sial cells . The RT-al cells .μM RA, 0.5 μM Pur , DMH1, SB, and CHIR for 6 days exhibited rapid changes in morphology. At this stage, the induced cells gathered centrally and formed rosettes  signaling pathway and RA . The NEProsettes , neural rosettes . Immunocrosettes . When werosettes .μM) and increased RA (0.5 μM) [To induce MNPs into functional MNs, we cultured MNPs in suspension with decreased Pur (0.1 (0.5 μM)  coupled (0.5 μM) , and LIM(0.5 μM)   , 20. RT-α-bungarotoxin (α-BTX), on the surface of the muscle cells [α-BTX and TuJ1 at sites on the axons of the UC005-MNs  marker, le cells  and coloC005-MNs , which iDirected differentiation of stem cells into MNs holds a great promise for the in vitro modeling of neurodegenerative diseases and cellular replacement therapies . Severalβ combined with inhibition of GSK-3β initiated the neutralization of iPSCs. After 7 days of induction, both B-iPSCs and urine-iPSCs differentiated into NEPs. The ratios of positive labeling for the neural precursor markers in NEPs derived from the three iPSC lines were all greater than 85%, and the ratio of the UC005-NEPs was slightly higher than those of the other two cell lines. During the subsequent 6 days, the presence of RA and Pur resulted in rapid changes in morphology, formation of rosettes, and upregulation of the MNP markers Olig2 and Pax6. The expression levels of the MNP markers in the UC005-MNPs and B-MNPs were significantly higher than those in the UE017-MNPs. With further differentiation in suspension, cells derived from all three iPSC lines congregated and formed MN spheres, which expressed the MN markers HB9 and Islet1. The rates of positive labeling for the precursor neural markers and MNP markers were significantly different among the cells from UC005, UE017, and B-iPSC, but the rates of positive labeling for the MN marker, HB9, were not statistically significant among the cells. For the further MN maturation, the combined actions of RA, Pur, and Cpd E resulted in the projection of long axons and expression of the mature MN marker, ChAT, in MNs derived from all three iPSC lines. When cocultured with muscle cells, urine-iPSC-derived MNs exhibited functional properties, including the projection of axons toward muscle cells, induction of muscle contractions, and NMJ formation. To further confirm our immunocytochemical observations, we used RT-PCR to analyze the mRNA expression levels of neuronal- and MN-specific markers. In all three iPSC lines, we detected expression of neuronal markers  and the MN-specific marker, HB9.We used Du's differentiation protocol , with slIn conclusion, the expression profiles of neural cell markers, as measured by immunocytochemistry and RT-PCR, demonstrated that urine-iPSCs were able to successfully differentiate into MNs. The formation of NMJs between MNs and muscle cells further indicated the functional properties of urine-MNs. In addition, the comparison of the ratios of positive labeling for MN markers demonstrated that the capacity of urine-iPSCs and B-iPSCs to differentiate into MNs is not significantly different. However, there are some important aspects that we did not investigate, such as the electrophysiological properties of induced MNs in vitro . Further


Mu suppression studies have been widely used to infer the activity of the human mirror neuron system (MNS) in a number of processes, ranging from action understanding, language, empathy and the development of autism spectrum disorders (ASDs). Although mu suppression is enjoying a resurgence of interest, it has a long history. This review aimed to revisit mu's past, and examine its recent use to investigate MNS involvement in language, social processes and ASDs. Mu suppression studies have largely failed to produce robust evidence for the role of the MNS in these domains. Several key potential shortcomings with the use and interpretation of mu suppression, documented in the older literature and highlighted by more recent reports, are explored here. Changes in mu power (i.e. the strength of the mu frequency band) have been used in recent years as a means to study the human mirror neuron system (MNS). Typically, investigations examining these changes take the form of mu suppression studies, in which the power of mu is compared between a baseline condition (which is not expected to excite mirror neuron activity) and an experimental condition .Figure Mu suppression studies have increased in number over the last decade. A recent meta-analysis surveyed 85 studies conducted since 1990 (including data from 1707 participants). This included only studies that examined mu rhythm activity in typical participants, and used an experimental paradigm that had an action observation condition or an action execution condition (or both)  also notThe human mu rhythm has a long history, going back to some of the earliest EEG experiments, long before the discovery of mirror neurons. One aim of this review is to give a brief overview of the history of mu, before its reconceptualization as a proxy for MNS engagement. Furthermore, given the widespread and increasing use of mu suppression to explore the existence, reactivity and potential function of the human MNS, it is pertinent to consider whether this method represents a sufficiently reliable and valid technique to infer MNS involvement. Thus, a second aim of this review was to outline problems in contemporary mu suppression studies. Finally, while traditionally mu suppression has been assessed in response to simple action observations, and the primary function of the MNS was regarded as action understanding –5, conte2.2.1.rythme rolandique en arceau, due to the waves' arch-like or wicket-like shape. Early observations were that these waves seemed to arise from the rolandic regions, at a rate of around 7–11 s−1 .The human mu rhythm was first described by the French scientist Henri Gastaut ,11, and et al.  decreases or disappears completely when the subject changes his position on his seat or when he readjusts his tonus. It also disappears when the subject identifies himself with an active person represented on the screen. This phenomenon is particularly interesting to study during a sequence of film showing a boxing match. A few seconds and, at times, less than a second after the appearance of the boxers all type of rolandic activity disappears in spite of the fact that the subject seems completely relaxed and that there is no noticeable change of posture. The relation between the blocking of the ‘arceau’ rhythm and the image of boxers in action is unquestionable. In the middle of this particular film strip, the camera is suddenly turned from the ring to the spectators in the hall for a few seconds. In many subjects the rhythm ‘en arceau’ reappears during this short period and vanishes again as the boxers reappear on the screen’ (p. 439).As well as reacting to participants' own movements, mu is suppressed by observing the movements of others. Reports of mu suppression during the observation of movements arose as scientists began to examine EEG responses to film projections. In a delightful paper by Gastaut & Bert , the autIn the 1980s, a team of Italian neuroscientists identified cells in the macaque brain that fired both when the animal performed an action and when it viewed an action being performed by another ,5. Theseper se )Finally, there is also a lack of studies that examine mu suppression in both perception and production within the same experiment, a test that seems crucial given that the crux of the motor theory of speech perception is the overlap between substrates supporting perception and production.In sum, it is debatable to what extent speech and language tasks lead to mu suppression independent of other potential confounds, or to what extent mu responses to speech stimuli align with what would be expected if the MNS plays a key role in speech and language processes. More confidence in findings could be achieved if some of the key studies could be replicated in a pre-registered design, in which hypothesis, methods and analytical approaches are decided upon prior to examination of the data, to prevent retrospective hypothesis fitting. These theories make clear, testable predictions and would seem well suited to this method of evidence-gathering .4.2.Following on from the original theories purporting that the MNS underpins action understanding, researchers have used mu suppression techniques to investigate the activation of the MNS in a number of related higher social processes, including empathy and theory of mind. Furthermore, there have been several investigations using mu to assess the role of the MNS in perceiving biological motion. Findings from mu suppression studies examining these skills are reviewed in turn here. Theories that link the MNS to empathy are highly similar to the theories that link the MNS to the separate but related construct of theory of mind. Therefore, these theories will be briefly discussed together, before examining the evidence for mu suppression during these different processes.4.2.1.One theory proposed to explain how we understand and empathize with the thoughts, feelings and beliefs of others is simulation theory. This theory posits that the same mental resources used in our own thinking and emotional responses are also used to understand the thoughts, feelings or beliefs of others, and stands in opposition to ‘theory’ theory—the notion that understanding others draws on empirical knowledge . Relatedet al.  biases… might be a manifestation of a more basic and general bias: perception–action-coupling for gross motor responses—the physiological process thought to be at the core of interpersonal sensitivity—might be impaired in response to disliked outgroups. Such a fundamental bias, would not only make it difficult to empathize with outgroup members' suffering, but also to understand their actions and intentions, potentially hampering smooth intergroup interactions and communication’ (p. 842). In a sample of 30 Caucasians, Gutsell et al. [r = 0.52). Gutsell & Inzlicht [Leading on from work on empathy, social scientists have also considered how mu suppression may be used to study the neural mechanisms for intergroup relations and prejudice. Drawing on the perception–action model of empathy, Gutsell et al.  hypothesl et al.  found siInzlicht  discuss 4.2.2.Despite considerable amount of research on empathy and mu suppression, only one study was found that used mu suppression to investigate MNS involvement in theory of mind. Pineda & Hecht  argued tPineda & Hecht  employedHowever, the results of this study are difficult to interpret. A direct comparison of the strength of mu suppression in the socio-cognitive and socio-perceptive tasks is not reported—so it is not possible to say whether socio-perceptive tasks result in greater mu suppression. Furthermore, the pattern of suppression across the tasks does not clearly demonstrate a difference between socio-cognitive and socio-perceptive tasks. For example, while significant suppression was seen during the emotion-matching task, significantly stronger suppression was seen during the race-matching task (though the authors interpret this as showing mirror neuron activity while participants make these race judgements). The authors also argue that the task with comics depicting physical causality with characters is actually a socio-perceptive task, as there are correlations between this and the emotion eye-matching task. Yet it is difficult to see why this physical-causality comic task would be underpinned by the MNS, yet the intention-reading comic task would not be. The key difference between these two tasks was the need to infer intention. This would seem, at face value, to be exactly the kind of skill the MNS was originally proposed to underlie. Pineda & Hecht  argue th4.2.3.Several studies have considered mu responses to biological point-light displays. These displays are image sequences created by marking the limb movements of moving bodies with lights. These stimuli offer a solution to the problems of presenting well-matched stimuli to investigate mu responses—social versus non-social stimuli typically differ on a number of basic perceptual factors, while point-light displays allow for a tighter control over such variables.Mu suppression to these displays has been used to argue that mirror neurons are involved in the processing of biological motion. In a study of 20 participants, Ulloa & Pineda  found siet al. [Indeed, other authors examining mu suppression to point-light displays have warned about potential confounding effects from occipital alpha and attentional differences in the different conditions. Perry et al.  examined4.2.4.The findings so far relating mu suppression to social processes are varied. Studies attempting to use mu suppression as an individual differences measure of the MNS seem particularly problematic. Few strong correlations have been observed , and it is unclear whether other theories not pertaining to mirror neurons could account for some of the findings. Certainly, many studies do not sufficiently consider the potential confounds of alpha or attentional effects in their designs  is logia priori characteristics of MNS activity [We believe the method of mu suppression can be refined, but it is also worth noting that it is not the sole technique that has been proposed as a means to study the properties of the human mirror system. Firstly, some have suggested that beta, rather than mu, may be an index of MNS engagement ,114,115,activity . TMS hasactivity ,117. Thiactivity . An alteactivity .et al. [We have focused our review largely on data collected from adults in mu suppression studies (with the exception of some of the studies of ASD); however, researchers have also used mu suppression studies with infant populations to try and address questions about the development of mirroring systems. Other researchers have reviewed mu suppression with infant populations, so this literature has not been re-examined here . Howeveret al.  outlineset al. –122. As and use this responsivity to suggest what stimuli the MNS is responsive to, but this logic is circular. Ideally, the field needs to agree what to expect the human MNS to respond to, examine whether mu suppression meets these expectations and reject it as a measure of the MNS if it does not meet them. Recent work on mu suppression suggests we need far more work establishing the reliability and validity of our measures, and agreeing on appropriate analysis pipelines, before we can use this approach with confidence to index activity of the human MNS [Furthermore, a comment not so much on the methodology of mu suppression studies but on their interpretation in wider social cognitive neuroscience: the impression one gets when reading the mu suppression literature is that theories about the function of the human MNS are sufficiently flexible to fit around whatever mu suppression results are obtained. Of course, given that theories about the MNS evolved and developed, we can expect to see mu suppression to stimuli beyond simple hands interacting with objects. But mu suppression has been demonstrated to viewing static buildings, sheet music and Rorschach ink blots ,105,123.uman MNS .While new data will be useful in making progress, this review also sought to reach back for evidence. Mu changes have long been considered to index motor-cortex engagement, well before mirror neurons exploded into the field of cognitive neuroscience. Considering mu's history, and how mu studies have changed over the last decade, should lead to reflection on how mu suppression should be conducted in the future. We hope that researchers will use this synthesis of the evidence to design and implement careful and considered mu suppression experiments in the future that can successfully rule out the confounds we and previous authors have outlined.