Ccna2)/cell division cycle 25C (Cdc25c) and Fermitin family homolog 2 (Fermt2) genes, respectively. However, it remains unclear how β-catenin targets different genes from stage to stage during myogenesis. Here, we show that the accessibility of β-catenin to the promoter region of its target genes is regulated by developmental stage-specific histone acetyltransferases (HATs), lysine acetyltransferase 2B (KAT2B), and cAMP-response element-binding protein (CREB)-binding protein (CBP). We found that KAT2B was specifically expressed at the myoblast proliferation stage and formed a complex with β-catenin to induce Ccna2/Cdc25c expression. On the other hand, CBP was specifically expressed during myoblast differentiation and formed a complex with β-catenin to induce Fermt2 expression. Our findings indicate that β-catenin efficiently accesses to its target gene’s promoters by forming a complex with developmental stage-specific acetyltransferases during myogenesis.Molecules involved in WNT/β-catenin signaling show spatiotemporal-specific expression and play vital roles in muscle development. Our previous study showed that WNT/β-catenin signaling promotes myoblast proliferation and differentiation through the regulation of the cyclin A2 ( Muscle precursor cells (aka satellite cells in adults) start to proliferate, at which stage they are referred to as myoblasts, and subsequently differentiate into myotubes2. WNT/β-catenin signaling contributes to both muscle cell proliferation and differentiation3. Upon binding WNT ligands to a Frizzled receptor (FZD) and the low-density lipoprotein receptor-related protein 5/6 (LRP5/6), β-catenin can stabilize and translocate from the cytoplasm into the nucleus. Nuclear β-catenin forms a complex with transcriptional co-activators, such as members of the T-cell factor (TCF)/lymphoid enhancer-binding factor 1 (LEF1) family, to bind the promoter regions of target genes4. By contrast, without WNT ligands, a destruction complex, which consists of AXIN, adenomatous polyposis coli (APC), and the serine-threonine kinase glycogen synthase kinase-3 (GSK3β), is activated and phosphorylates β-catenin, leading to its degradation through the ubiquitin-proteasome system5. While WNT/β-catenin signaling plays a crucial role in both proliferation and differentiation during myogenesis3, it remains unclear how β-catenin controls the expression of genes for either proliferation or differentiation.Skeletal muscle development and regeneration are controlled through complex regulatory processes7. Among them, histone (H) acetylation at lysine (K) residues is crucial for the activation of gene transcription8. The bromodomain, a small protein domain of histone acetyl transferase (HAT), functions in the linking of protein complexes to acetylated nucleosomes, thereby controlling the transcription of target genes through chromatin structure changes. For example, H3K9 and H3K27 acetylation (H3K9Ac and H3K27Ac) activates promoter/enhancer regions of genes that are then transcribed9. Lysine acetyltransferase 2B (KAT2B) [aka p300/CBP-associated factor (PCAF)] is a transcriptional coactivator that works as a HAT for H3K9, and as an acetyl-lysine reader through its conserved bromodomain located at C-terminal of the HAT domain10. By contrast, CREB-binding protein (CBP) specifically acetylates H3K27 through the HAT and E1A binding domains9.Post-translational modifications  of core histone proteins change chromatin conformationIn this study, we examined the molecular mechanism of how WNT/β-catenin signaling regulates its target genes in a temporal-specific manner. We found that the β-catenin complex incorporates either KAT2B or CBP in a developmental stage-specific manner, regulating the accessibility of β-catenin to the target gene’s promoters.11. In our previous study, we found that WNT/β-catenin signaling regulates myoblast proliferation and early differentiation (myoblast fusion) through gene regulation of cyclin A2 (Ccna2), cell division cycle 25c (Cdc25c), and Fermitin family homolog 2 (Fermt2) in C2C12 cells3. However, it was still unclear how the expression of these genes was regulated by WNT/β-catenin signaling in a proliferation and differentiation stage-specific manner. The gene expression of Ccna2 and Cdc25c, crucial for cell cycle progression, was much higher in the proliferation stage than in the differentiation stage and was downregulated by a WNT/β-catenin signaling inhibitor, IWR1-endo, while Ccnd3 expression was not altered by IWR1-endo  in C2C12 cells during cell proliferation and muscle differentiation. We found that Kat2b and Cbp were highly and specifically expressed during cell proliferation and muscle differentiation, respectively  and H3K27Ac in the promoter regions of Ccna2, Cdc25c and Fermt2. H3K9Ac was markedly increased in the proliferation stage on the Ccna2 and Cdc25c promoters, compared to the muscle differentiation stage  at the proliferation and differentiation stages. The interaction between β-catenin and KAT2B was increased in the proliferation stage. By contrast, the interaction between β-catenin and CBP was increased in the differentiation stage Fig. . FurtherCcna2, Cdc25c and Fermt2, we performed knockdown experiments for Ccna2, Cdc25c and Fermt2 in the cell proliferation and differentiation stages. Kat2b knockdown decreased the expression of Ccna2 and Cdc25c in the cell proliferation stage  compared to controls  are specifically expressed in skeletal muscle and differentially regulate the gene expression of myogenic factorsIn summary, our results indicate that epigenetic factors play crucial roles in the regulation of WNT/β-catenin signaling target gene by directly interacting with β-catenin. Our findings on the epigenetic regulation of WNT/β-catenin signaling offer several intriguing possibilities into the potential for therapeutic interventions to stimulate effective skeletal muscle regeneration following muscle trauma or atrophy.2 in a humidified incubator, as previously described3. Myogenic differentiation was induced with muscle differentiation medium  for 48 hours with/without IWR1-endo , a WNT/β-catenin signaling inhibitor, at the indicated concentration, as previously described3. The small interfering RNA (siRNA) knockdown for Kat2b, Cbp and Ctnnb1 (Santa Cruz) was performed as described previously3. Bromodeoxyuridine (BrdU) incorporation assays were performed using cells treated with control or Kat2b siRNA knockdown for 24 hours and control or Cbp siRNA knockdown for 48 hours. Incorporated BrdU was stained with a rat monoclonal antibody against BrdU (Abcam), as described previously3. A total of 10 fields, randomly selected from three independent experiments, were used for the quantification of BrdU-positive cells. The antibodies used in this study are shown in Supplementary Table C2C12 cells, a murine skeletal muscle cell line, were obtained from the American Type Culture Collection . C2C12 cells were cultured in growth medium  for 24 hours at 37 °C and 5% COKat2b siRNA or control siRNA for 24 hours (n = 3 per group), were extracted with a QIAshredder and RNeasy mini extraction kit (QIAGEN), as described previously39. For the myoblast differentiation stage, total RNAs isolated from C2C12 cells cultured under differentiation medium with 1 µM IWR1-endo or control vehicle for two days (n = 3 per group) and Cbp siRNA or control siRNA for two days (n = 3 per group) were dissected with the QIAshredder and RNeasy mini extraction kit (QIAGEN), as described previously40. The PCR primer pairs used for further specific analysis are shown in Supplemental Table For the cell proliferation stage, total RNAs isolated from C2C12 cells, treated with IWR1-endo or control vehicle for 24 hours (n = 3 per group) and 41. The putative LEF1 target sites of the genes in the immune complexes were detected by PCR using the following primers: Ccna2 gene, 5′-TGGTGTTGCAGATCTACCGT-3′ and 5′-TCTGCTAACAAAATGGCAATGC-3′ (−2364 to −2154); Cdc25c gene, 5′-TTCATCGGTCTCAGCTTCCC-3′ and 5′-AGTCACCACTGAGCCTTGTC-3′ (−3164 to −3029); and Fermt2 gene, 5′-TAGAGGTTCTAGCGGGGGTT-3′ and 5′-TTCAGGCCTTGGCTTTGAGT-3′ (−3138 to −2949). The positions of the PCR fragments correspond to NCBI mouse genome Build 38 (mm10).Cell extracts were incubated with either active β-catenin, H3K9Ac, or H3K27Ac antibody  overnight at 4 °C, followed by precipitation with magnetic beads. Washing and elution of the immune complexes, as well as precipitation of DNA, were performed according to standard procedures, as described previously41. Mouse monoclonal antibodies against β-catenin (Abcam), KAT2B (Santa Cruz) and CBP (Abcam) were used for IP assays. Immunoblots were performed using rabbit polyclonal antibodies against active β-catenin, KAT2B, and CBP , as described previously42.Nuclear extracts were prepared for IP using a Nuclear Extraction Kit (Thermo Scientific). IP assays were performed as described previously3, using the following antibodies: mouse monoclonal antibody against active β-catenin (Millipore), rabbit polyclonal antibodies against KAT2B  and CBP  and mouse monoclonal antibodies against myosin heavy chain (MYH) (Sigam-Aldrich) and MyoD1 (Thermofisher). Confocal images were obtained with a confocal microscope . Fluorescent images of MYH immunostaining were captured by an inverted fluorescence microscope . A total of 10 fields, randomly selected from three independent experiments, were used for the quantification of the length of myotubes and fusion index.Immunofluorescence analysis was performed, as described previouslyt tests were applied for statistical analysis. A p value ≤ 0.05 was considered statistically significant. For all graphs, data are represented as mean ± standard deviation (SD).Two-tailed student’s Supplemental Information


Wingless-type mouse mammary tumor virus (MMTV) integration site (Wnt) signaling is one of the most critical pathways in developing and adult tissues. In the brain, Wnt signaling contributes to different neurodevelopmental aspects ranging from differentiation to axonal extension, synapse formation, neurogenesis, and neuroprotection. Canonical Wnt signaling is mediated mainly by the multifunctional β-catenin protein which is a potent co-activator of transcription factors such as lymphoid enhancer factor (LEF) and T-cell factor (TCF). Accumulating evidence points to dysregulation of Wnt/β-catenin signaling in major neurodegenerative disorders. This review highlights a Wnt/β-catenin/glial connection in Parkinson’s disease (PD), the most common movement disorder characterized by the selective death of midbrain dopaminergic (mDAergic) neuronal cell bodies in the subtantia nigra pars compacta (SNpc) and gliosis. Major findings of the last decade document that Wnt/β-catenin signaling in partnership with glial cells is critically involved in each step and at every level in the regulation of nigrostriatal DAergic neuronal health, protection, and regeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD, focusing on Wnt/β-catenin signaling to boost a full neurorestorative program in PD. Parkinson’s disease (PD) is the most common movement disorder, and the second most common aging-related neurodegenerative disease after Alzheimer’s disease. The selective and progressive degeneration of midbrain dopaminergic (mDAergic) neurons of the substantia nigra pars compacta (SNpc) and their projections into the caudate–putamen (striatum) leading to substantial decrease in dopamine levels, the accumulation of pathological α-synuclein, which is a major component of Lewy bodies (LBs), and gliosis as the major hallmarks of PD ,2. As thSNCA, LRRK2, and VPS35, and autosomal recessive mutations in PINK1, DJ-1, and Parkin cause the disease with high penetrance -DA incorporation; MPP+ application to astrocyte–neuron cultures resulted in a significant degree of neuroprotection  dopamine incorporation, as compared to neuronal cultures treated with control siRNA, where Wnt1 treatment afforded full TH+ neuroprotection [Hence, in three PD cellular models  in face of exacerbated levels of inflammatory and oxidative stress genes, including IL-1β, TNF-α, IL-6, iNOS2, nuclear factor kappa B (NF-κB), and the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase, Phox, that accompanied the failure of nigrostriatal DAergic self-repair [Remarkably, aging and MPTP exposure further amplified Wnt/β-catenin down-modulation in SNpc tissues of 16–20-month-old mice, since the endogenous Wnt-antagonists, f-repair .+/− mice), and characterized by the age-dependent and slowly progressing degeneration of the mDAergic neurons [+/Wnt1 VM activated a gene cascade that protected these genetically affected En1 heterozygote (En1+/−) neurons from their premature degeneration in the adult mouse VM. Hence, the direct Wnt1/β-catenin signaling targets lymphoid enhancer-binding factor 1 (Lef1), LIM homeobox transcription factor 1 (Lmx1a), fibroblast growth factor 20 (Fgf20), and Dkk3, as well as the indirect targets pituitary homeobox 3  and brain-derived neurotrophic factor , playing decisive roles in mDA neurodevelopment, were significantly upregulated [Of particular mention, the key neuroprotective role of Wnt1 in the aged midbrain was highlighted in the recent study of Zhang and colleagues in 2015 , in mice neurons . Here, tegulated . The autegulated .Dkk1 resulted in a significant degeneration of DAergic cortico-striatal excitatory synapses in striatum and a decrease in glutamate release from cortico-striatal afferents [Dkk1 in the hippocampus exhibit synapse loss, impaired long-term potentiation, enhanced long-term depression, and learning and memory alterations [Dkk1 can be reverted by Wnt agonists or by the inhibition of GSK-3β, thereby indicating that “the endogenous Wnt proteins are critical to maintain synaptic connections in the adult brain” [Furthermore, a major role of a physiological Wnt tone for synaptic maintenance and function in the adult striatum was recently uncovered . The stufferents . Moreoveerations ,136. Nott brain” .Taken together, these results suggest that, in PD brain, an early downregulation of Wnt signaling starting by middle age might predispose the vulnerable nigrostriatal DAergic neurons to dysfunction and/or death. In synergy with the aging process, different risk factors, including inflammation and MPTP exposure, may act in concert to impair Wnt signaling both inside and ouside the neurogenic niches, with harmful effects for nigrostriatal functionality and neurogenesis. Importantly, the potential exists to revert some of these age-dependent changes, which is discussed in In the adult brain, neural progenitors in neurogenic areas such as the SVZ and the subgranular zone (SGZ) of the hippocampus are in intimate contact with astrocytes which helps generate an instructive “niche” that promotes neurogenesis ,139,140.Interestingly, in vivo studies in young adult mice revealed that MPTP-induced SNpc neuron death promoted a remarkable astrocyte-dependent remodeling within the Aq-PVR niche . FascinaCanonical Wnt/β-catenin signaling reporter mice are strains of transgenic mice with a LacZ transgene controlled by TCF/LEF consensus DNA-binding elements and a minimal promoter . The estAs, with the aging process, Wnt signaling in the brain declines, this results in the impairment of Wnt-mediated self-protective, neuroreparative, and neurorestorative DAergic mechanisms. Of special interest, in the aged PD mouse model, the changing properties of the midbrain Aq microenvironment resulted in reduced DAergic neurogenic potential of Aq-NSCs via a loss of astrocytic Wnt1 and a failure of Wnt/β-catenin signaling activation . This efWnt1 expression or under Wnt/β-catenin activation regimens, such as GSK-3β antagonism, leading to DA neuron formation, in vitro [Ex vivo and in vitro studies coupled to different co-coculture paradigms and a panel of experimental conditions next indicated that both glial age and a decline of glial-derived factors, including Wnt1, were responsible for impaired NSC neurogenic potential within the SVZ ,55 and Ain vitro . In vivoin vitro .Wnt1. In particular, these NSC-derived astrocytes promoted a remarkable time-dependent endogenous nigrostriatal DAergic neurorepair [Finally, the unique role of astroglial-derived Wnt1 in aged MPTP-injured mice was further highlighted very recently, following NSC transplantation in the injured SNpc, which promoted a remarkable time-dependent nigrostriatal DAergic neurorescue/repair . Here, Srorepair . Astrocyrorepair .Taken together, these results suggest the potential to restore mDAergic neuron functionality by activating Wnt/β-catenin signaling via both pharmacological and cellular approaches aimed at rescuing the imperiled mDAergic neurons, downmodulating the exacerbated microglial reaction, and via the activation and/or the recruitment of endogenous mDAergic progenitors ,80.In the last few years, an increasing number of pharmacological and immunomodulatory agents affording neuroprotection were recognized as activators of the canonical Wnt/β-catenin signaling pathway. Different studies focused on the neuroprotective capacity of Wnt1-agonists and pharmacological inhibitors of GSK-3β. Hence, in 2013, Wei et al.  supporteOther studies indicated the potential of Wnt1-like agonists, such as Wnt1 inducible signaling pathway protein 1 (WISP1), a downstream target in the Wnt1 pathway, to block neurodegeneration ,158,159.The interrelationships among inflammatory, survival, and Wnt/β-catenin signaling cascades also uncovered a complete regulatory loop impacting in both neurogenesis and neuronal outcome upon injury, protecting mDAergic neurons from loss in the MPTP mice model of PD through inflammatory inhibition, via activation of PI3K/phosphorylated (p)-Akt and Wnt1/Fzd1/β-catenin cell signaling pathways ,160,161,Together, these and other findings of the last few years support the indication of Wnt/β-catenin signaling as a critical final common pathway for mDAergic neurorescue, with an increasing interest in its therapeutic targeting ,43,106. A central challenge in the field of neurodegenerative disorders is developing therapeutic strategies that boost neuroprotection and neurorepair, and that help endogenous regenerative programs in the injured brain. This work summarized several lines of evidences supporting a key role for the canonical Wnt/Fzd-1/β-catenin pathway contributing to the maintainance of mDAergic neuron survival and functionality within the adult intact and PD midbrain. Accumulating data from the studies of genes mutated in PD underscore the pivotal role of Wnt signaling pathways for the function of mDAergic neurons in health and disease states. New potential associations between the Wnt pathway and mitochondrial dynamics, lysosomal biogenesis, vesicle trafficking, apoptosis, autophagy, immunoregulation, and cell cycle, affecting key functions in neuroprogenitors, post-mitotic neurons, and glial cells are starting to be unveiled.Notably, Wnt1-induced neuroprotection is closely related to the astroglial response to oxidative stress and inflammation upon injury, and requires Fzd-1 receptor and β-catenin stabilization coupled to GSK-3β inhibition to promote mDAergic neuron survival. Reactive astrocytes upregulate Wnt/β-catenin signaling. This astrocyte-dependent property might help in mitigating excessive inflammation as observed during aging and neurodegeneration. Importantly, Wnt signaling at the neuroimmune interface plays a pivotal role in the regulation of neuroprogenitors, post-mitotic neurons, and microglial cell functions in PD. Also, pharmacological manipulation of microglial oxidative and nitrosative status, either in vitro or in vivo, can activate a beneficial Wnt/β-catenin signaling, thus affording neuroprotection and inducing a successful neurogenesis rescue. Therefore, modulating these astroglia changes may represent a powerful ground for novel therapeutic intervention strategies to prevent, delay progression, and/or ameliorate pathology.Together, these studies suggest that Wnt-induced pathways are required for the regulation of a panel of processes linked to mDAergic neuroprotection, neurorepair, and regeneration after injury, and include the re-expression of genetic neurodevelopmental programs for DA neuroprogenitor acquisition of the mature mDAergic phenotype, DA neuron formation, and migration, as well as their survival and maintenance within a hostile microenvironment thanks to Wnt’s immunomodulatory properties.Given that the activity of Wnt signaling in the brain declines with the aging process, this results in the inhibition of Wnt-mediated self-protective, neuroreparative/neurorestorative, and immunomodulatory mDAergic programs. Because Wnt/β-catenin signaling controls the expression of a wide panel of direct and indirect target genes, mis-regulation of this signaling cascade may be involved in various age-dependent diseases associated with impaired neurogenesis, including PD. Notably, the signaling mechanisms contributing to neuronal death and NSC impairment in the aged PD brain target the Wnt/β-catenin signaling pathway. Coupled to the increasing evidence on the key role of Wnt/β-catenin signaling cascades in neurodevelopment, neurodegeneration, and regeneration, developing targeted in situ pharmacological interventions/cell manipulations that boost the inherent Wnt-dependent regenerative potential have implications for both restorative and regenerative approaches in PD.Notably, given the multitude of roles of Wnt/β-catenin signaling in the control of tissue homeostasis and disease, there is an increasing interest in targeting this pathway. From a therapeutic viewpoint, the Wnt signaling pathways presents several challenges to the development of a targeted neuroprotective drug, and further studies are still needed to elucidate the complex Wnt signaling circuitry. For example, the Wnt pathway itself is protective in different tissues, but the receptor context may influence the regulation of canonical Wnt signaling by agonist or antagonists. In addition, the cellular context, the presence of gene mutations, and the interaction with a panel of environmental risk factors, such as aging and inflammation, may finally mediate the tissue-/cell-specific response.As a whole, the understanding of the intricate signaling networks of Wnt/β-catenin signaling is critical for the identification of new potential therapeutic targets and the development of pharmacologic and cellular approaches for neurodegenerative diseases, including PD.


Chrysosplenetin is an O-methylated flavonol compound isolated from the plant Chamomilla recutita and Laggera pterodonta. The aim of our research is to evaluate the function of Chrysosplenetin on osteogenesis of human-derived bone marrow stromal cells (hBMSCs) and inhibition of estrogen deficiency-induced osteoporosis via the Wnt/β-catenin signaling pathway.Chrysosplenetin in the absence or presence of Wnt inhibitor dickkopf-related protein 1 (DKK1) or bone morphogenetic protein 2 (BMP2) antagonist Noggin. RT-qPCR is taken to identify the genetic expression of target genes of Wnt/β-catenin pathway and osteoblast-specific markers. The situation of β-catenin is measured by western blot and immunofluorescence staining. An ovariectomized (OVX) mouse model is set up to detect the bone loss suppression by injecting Chrysosplenetin. Micro-CT and histological assay are performed to evaluate the protection of bone matrix and osteoblast number. Serum markers related with osteogenesis are detected by ELISA.hBMSCs are cultured and treated by Chrysosplenetin time-dependently promoted proliferation and osteoblastogenesis of hBMSCs reaching its maximal effects at a concentration of 10 μM. The expressions of target genes of Wnt/β-catenin pathway and osteoblast-specific marker genes are enhanced by Chrysosplenetin treatment. Furthermore, the phosphorylation of β-catenin is decreased, and nuclear translocation of β-catenin is promoted by Chrysosplenetin. Osteogenesis effects mentioned above are founded to be blocked by DKK1 or BMP2 antagonist Noggin. In vivo study reveals that Chrysosplenetin prevents estrogen deficiency-induced bone loss in OVX mice detected by Micro-CT, histological analysis, and ELISA.In the present study, it is found that Chrysosplenetin improves osteoblastogenesis of hBMSCs and osteogenesis in estrogen deficiency-induced bone loss by regulating Wnt/β-catenin pathway.Our study demonstrates that The online version of this article (10.1186/s13287-019-1375-x) contains supplementary material, which is available to authorized users. Postmenopausal osteoporosis (PO) is characterized by bone mineral reduced and architectural deterioration in the skeletal system due to deficiency of estrogen , 2. BasiIt is highlighted that Wnt/β-catenin signaling pathway plays a great role in the bone homeostasis , 6. Wnt Chrysosplenetin is an active O-methylated flavonol extracted from Chamomilla recutita and Laggera pterodonta  for anti-inflammation and neuro disease [Chrysosplenetin on osteoblastogenesis of BMSCs.nta Fig. . Previoug cancer  and antig cancer . It is rrization . Chrysos disease . HoweverChrysosplenetin on osteoblastogenesis of hBMSCs in vitro. It is indicated that Chrysosplenetin promotes osteoblastic differentiation of hBMSCs and increases canonical Wnt/β-catenin signaling pathway. Furthermore, an ovariectomized (OVX) mouse model was set up to investigate the physiological efficiency of Chrysosplenetin on PO in vivo. The result suggests that estrogen deficiency-induced bone loss is significantly inhibited by Chrysosplenetin treatment. Our study has revealed that Chrysosplenetin may improve osteoblastogenesis of BMSCs via Wnt/β-catenin signaling pathway and prevent estrogen deficiency-induced bone loss.In our study, we innovatively identify the effects of Chrysosplenetin (purify ≥ 98%) was obtained from the TransMIT Project Division for Plant Metabolites and Chemicals  and dissolved in dimethyl sulfoxide (DMSO) purchased from Sigma-Aldrich . It was firstly dissolved as a primary concentration of 10 mM and then diluted to final concentrations as required in the culture medium. Low-glucose Dulbecco minimum essential medium (LG-DMEM), penicillin-streptomycin, and fetal bovine serum (FBS) were ordered from Thermo Fisher Scientific . Human Dickkopf-related protein 1 (DKK1) and Noggin recombinant protein were obtained from Sigma-Aldrich  and dissolved in bovine serum albumin (BSA) . β-Catenin antibody, phosphorylation-β-catenin (p- β-catenin), and β-actin antibody were purchased from Cell Signaling Technology . All antibodies were used at the concentrations recommended by the supplier at 1:1000. Second antibodies were purchased from Thermo Fisher Scientific . Mineral deposition of osteoblast was detected using the Alizarin Red Staining Kit . Alkaline Phosphatase (ALP) Activity Fluorometric Assay Kit was purchased from Abcam . ELISA kit of Ot/Bgp, Balp, and Ct were obtained from R&D Company . Human bone marrow stromal cells (hBMSCs) were obtained from Cyagen Biosciences Inc. .2 at 37 °C. The medium was replaced for every 3 days until the flask was confluence and filled with cells. Then, hBMSCs were digested and passaged to the next generation for up to three times. The hBMSC suspension for each passage was grouped. The cell-surface antigen  and correspondence isotype control) of the hBMSCs was identified. After that, hBMSCs were washed twice with phosphate-buffered saline (PBS) and fixed in 1.0% paraformaldehyde (PFA). Flow cytometry was undertaken for the evaluation of surface antigen. Subsequently, hBMSCs from 2 and 3 passages were used for the following experiment in our test.hBMSC suspension was seeded into a culture flask and cultured in a completed DMEM medium . The monolayer culture of hBMSCs was kept in an incubator with a condition as 5% CO4 cells/well) for overnight incubation. Chrysosplenetin  was then added to cells in the presence of osteogenic induction medium . Plates with cells were incubated at 37 °C for 1, 2, 3, 7, and 14 days. MTT assay was measured to identify the effect of Chrysosplenetin on the proliferation of hBMSCs. 3--2,5-diphenyltetrazolium bromide was added into each well for further 3 h. Then, the formazan crystals were dissolved in DMSO. The absorbance was evaluated at 490-nm wavelength to assess the viability of hBMSCs.hBMSC suspension were plated in 96-well plates  for 14 days. Second, to determine the effects of Chrysosplenetin, Noggin, and DKK1 on the osteogenic differentiation of hBMSCs, hBMSCs with OIM were treated by Chrysosplenetin at an optimal concentration in the presence or absence of Noggin (10 μg/ml) or DKK1 (0.5 μg/ml).In our test, there are two strategies for the following evaluation. First, in order to evaluate the effect as well as optimal concentration of 6 per well for osteoblastogenesis. After osteogenic induction in strategy one, alizarin red staining and ALP activity assay were performed to identify bone mineral of cells according to the manufacturers’ introduction. Specifically, cells were cultured in varying concentrations of Chrysosplenetin for 14 days and fixed with 4% PFA for 30 min, followed by PBS washing. Alizarin red staining solution was added into the wells for 5 min. The cells were viewed using a 450 fluorescent inverted phase-contrast microscope .hBMSCs were seeded and cultured in the 6-well plates at a density of 1 × 10As for ALP activity assay, hBMSCs were modulated same as what in alizarin red staining test. Then, cell lysates were collected in a test tube with alkaline solution and subjected to ALP activity analysis by a fluorometric detection kit. The absorbance set up as 450 nm was measured using a microplate reader .6 cells per well. In strategy one, the mRNAs of osteoblastic genes including Runt Related Transcription Factor 2 (RUNX2), Osteocalcin (BGLAP), β-catenin (CTNNB1), and Bone Morphogenetic Protein 2 (BMP2) were detected. In strategy two, the mRNAs of osteogenic genes, including RUNX2, Distal-less Homeobox 5 (DLX5), Osteopontin (SPP1), Collagen type I (COL1), BGLAP, and BMP2, and Wnt/β-catenin target genes, including CTNNB1, Transcription Factor 7 (TCF7), Lymphoid Enhancer Binding Factor 1 (LEF1), MYC (C-MYC), cyclin D (CCND1), and c-JUN (JUN), were identified for analysis. PCR reactions using specific primers of the genes were seen in the Additional file For real-time PCR, hBMSCs were seeded in 6-well plates at a density of 1 × 10−∆∆CT) and normalized against GAPDH. Reaction products were separated using agarose gel electrophoresis and visualized on an Image-quant LAS 4000 .hBMSCs after osteogenic induction mentioned above were lysed. Then, the total RNA was isolated from the hBMSCs by adding Trizol reagent  in accordance with the product instruction. Concentration of RNA was determined by Thermo Scientific Microplate Reader , and single-stranded cDNA was reverse transcribed. qPCR reactions were performed in a ViiA 7 Real-time PCR system . Polymerase chain reaction amplification of specific sequences was cycled in the following condition: 94 °C for 5 min, followed by 30 cycles of 94 °C (40 s), 60 °C, (40 s), and 72 °C (40 s), and a final dissociation of 30 s at 72 °C. Data was showed as Ct (25 cells/well) and pre-treated with OIM for 3, 7 and 14 days. hBMSCs in the Chrysosplenetin test group were treated with or without Noggin or DKK1. After that, cells were lysed with RIPA Lysis buffer  for protein extraction and pelleted at 14,000×g for 5 min. Proteins were identified by Bradford Protein Assay Kit . Samples then were separated using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer electrophoresis  and transferred onto nitrocellulose membranes . The membrane was blocked in 5% skim milk diluted in 1× TBS-Tween , 150 mM NaCl, 0.05% Tween-20) for 1 h at room temperature, then probed with specific antibodies, including p-β-catenin and β-catenin antibodies (diluted as 1:1000) at 4 °C for overnight in a shaking manner. The membrane was washed in TBST and incubated in appropriate horseradish peroxidase-conjugated secondary antibodies (1:5000). The membrane was then developed using the ECL reagents . Band intensity was normalized against β-actin. Finally, an Image quant LAS 4000  was used to figure out the membranes.Fresh hBMSCs were seeded into 12-well plates  for 30 min. After three washes of PBS with 5 min for each time, cells were incubated for 1 h with primary anti-β-catenin antibodies at 1:100 dilution at room temperature. After the first-round incubation and three washes of PBS again, cells were incubated for 30 min with fluorescein isothiocyanate (FITC)-linked goat anti-rabbit IgG conjugated at 1:100 dilution. Finally, cell samples were washed for 40-6-Diamidino-2-phenylindole  staining to highlight nuclei of the cells. Then, the cellular samples were observed by using a confocal microscope , and captured images were acquired using the Image-Pro Plus 6.0 software .Chrysosplenetin injection). The mice were kept individually in cages with standard chow and water support and a half-day light/dark cycle. The OVX group and test group were anesthetized and undertaken an ovariectomy inducing estrogen deficiency, whereas the sham operation was performed in the sham group as a normal control. Each ovary in the OVX group and test group was completely removed with its capsule and part of the oviduct. All of groups had 1 week for recovery and incision healing. After that, mice in the test group were given an intraperitoneal injection of Chrysosplenetin at 3 mg/kg for every 2 days, instead of 1% DMSO in the sham and OVX groups. At 6 weeks post-treatment, the mice were euthanized. The femur was removed and fixed in 4% PFA for 24 h. The protocols involving animal model were approved by Guangzhou University of Chinese Medicine Institutional Animal Ethics Committee.Totally, 18 specific pathogen-free C57BL/6 J female mice (7 weeks of age) were randomly divided into three groups and six mice for per group  for scanning. The parameters of scanning setting were as follows: 70 kV, 200uA, 10-μm Al filter, 300-ms exposure, pixel size 8.89 μm, 2 frame averaging, and 0.4-degree rotation step through 180°. A resolution as high as 9 μm was obtained in scanning CT images, and 3D reconstruction was established. We determined the proximal femur as our region of interest (ROI), to be specific, volume starting 0.5 mm from the bottom of the growth plate for a 1-mm distance. The several parameters of trabecular bone within this volume including volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were manually identified by a constant threshold.By following the micro-CT analysis, bone samples were fixed with 4% PFA and then decalcify later. Samples were cut and prepared as the sequential 5-mm-thick sections stained using hematoxylin and eosin (H&E). As for immunohistochemistry, the sections were de-paraffinized and rehydrated and incubated with primary antibody against (1:600) overnight at 4 °C. For detection, sections were incubated with HRP-conjugated secondary antibody for 60 min, followed by the addition of liquid DAB substrate . Sections were counterstained with hematoxylin and were dehydrated and mounted with fast-drying mounting media . Sections were scanned using Aperio Scanscope , and bone histomorphometric analyses were performed using BIOQUANT OSTEO software .Ot/Bgp), Bone Alkaline Phosphatase , and Calcitonin (Ct) was assessed using ELISA kit according to the manufacturers’ introduction.Serum was isolated from the abdominal aorta of mice in each group, and the level of Osteocalcin/Bone Gla Protein  of the values obtained from three repeated experiments. All the experiments were repeated at least three times and presented as the average of triplicate independent experiments or the data from a representative experiment. Statistical significance was determined by Student’s To identify the characteristic and phenotypic of hBMSCs, primary cell culture and surface marker detection were performed. hBMSCs were expanded in primary culture and passaged for 3 to 5 times. In primary phase, attached hBMSCs obtained a 90% confluence at days 7. hBMSCs at passages 1 and 2 reached 90% confluence at day 3  at day 14 of culture  in the nucleus of the hBMSCs (stained with blue color) was observed in the control group. By adding Chrysosplenetin (10 μM) into hBMSCs, obviously enhanced translocation of β-catenin was noticed in the nucleus. However, pretreatment with Noggin and DKK1 enable to block the Chrysosplenetin significantly by reducing the nuclear translocation with low level of β-catenin in merged images  or DMSO only for control. There was no adverse event discovered during the whole procedure. In micro-CT assessment, the result revealed that Chrysosplenetin significantly reduced the bone loss associated with ovariectomy, as shown by upregulations of BV/TV, Tb.N, and Tb.Th in test mice. Trabecular separation (Tb. Sp) was prevented in the test group when compared with the OVX control group  (osteoblasts number/bone perimeter) and Ob.S/BS(%) (osteoblast surfaces/bone surface) as well as Bgalp expression in bone tissue , Osteocalcin (BGLAP), β-catenin (CTNNB1), Bone Morphogenetic Protein 2 (BMP2) were detected. In Strategy two, the mRNAs of osteogenic genes, including RUNX2, Distal-less Homeobox 5 (DLX5), Osteopontin (SPP1), Collagen type I (COL1), BGLAP and BMP2, and Wnt/β-catenin target genes, including CTNNB1, Transcription Factor 7 (TCF7), Lymphoid Enhancer Binding Factor 1 (LEF1), MYC (C-MYC), cyclin D (CCND1) and c-JUN (JUN). (DOCX 15 kb)PCR reactions used specific primers of the genes. The table provides the information of PCR reactions used specific primers of mRNAs detected in our research, including Runt Related Transcription Factor 2 (


Microneedles (MNs) have been established as promising medical devices as they are minimally invasive, cause less pain, and can be utilized for self-administration of drugs by patients. There has been rapid development in MNs for transdermal monitoring and diagnostic systems, following the active research on fabrication methods and applications for drug delivery. In this paper, recent investigations on bio-sensing using MNs are reviewed in terms of the applicability to continuous glucose monitoring system (CGMS), which is one of the main research focuses of medical engineering technologies. The trend of the functionalized MNs can be categorized as follows: (i) as a sensing probe, and (ii) as a biological fluid collector. MNs as in vivo sensors are mainly integrated or coated with conductive materials to have the function as electrodes. MNs as fluid collectors are given a certain geometrical design, such as a hollow and porous structure aided by a capillary action or negative pressure, to extract the interstitial fluids or blood for ex vivo analysis. For realization of CGMS with MNs, a long-term accurate measurement by the MN-based sensing probe or a fluidic connection between the MN-based fluid collector and the existing microfluidic measurement systems should be investigated. Diagnostic systems using bio-sensing technologies have been the main focus of research for the last few decades owing to the high demand for more rapid, precise, and easy-to-use applications for the well-being of society. One of the aims in the development of recent diagnostic devices is to achieve a shorter time period of analysis with a reduced amount of the required biological samples by microfluidic and electrochemical components realized by new fabrication technologies, such as the CMOS and MEMS. However, in spite of the advantages of such diagnostic systems, called micro total analysis systems (pTASs), the methodologies of body fluid sampling is limited to conventional methods such as hypodermic needles.Conventional needles have several disadvantages such as the associated unavoidable pain, and requirement of a high level of skilled training of the medical professionals, even though they extract too much amount of the body fluids for pTASs. Hence, microneedles (MNs) have attracted significant attention as next-generation devices to access biological tissues through the skin without pain due to their microsized dimension, e.g. typically a length of less 1 mm and diameter of less 500 pm. Subsequent to the reports of MNs being novel tools to penetrate the skin and to deliver drugs and vaccines with a minimal invasion and pain, a wide range of research has focused on the methods of their fabrication, supported by micromachining technologies and the applications for transdermal, self-administration of drugs .In addition to the medical drug delivery, MNs, e.g., dissolvable MN patches, are also well known as commercialized cosmetic products for skin treatments , 14 nowa+ and Na+ ions, nitrogen oxide, and glucose, the analysis, as well as that of blood . I. I66]. IAn alternative way to access biological information is to extract body fluids through the micro pores punctured by the MNs. In this category, the MNs are defined as biological fluid collectors creating a transdermal fluidic path at the interface between the inner tissue including the dermis and the outer part of the skin. The MNs collecting biological fluids such as blood and ISF have attracted the interest of researchers as they have the potential to be utilized in the existing ex vivo diagnostic systems including pTAS devices. However, it is necessary for the MN-based collector to collect at least a few pL of liquid for a reliable measurement of the analytes –74, wherResearch on the application of hollow MNs for the collection of biological fluid was initiated earlier than that of other types of MNs, probably due to their suitability to the sampling of a sufficient amount of the biological fluid in a manner similar to conventional hypodermic needles. The hollow MNs extract ISF or blood into their bores by a capillary action or external negative pressure. As the hollow MNs were put to use as micro injection needles for localized drug delivery into the skin at the initial stage of the development of research in MNs , 75, thex-immobilized paper channel. The glucose level is observed by comparing the colorimetric change of the paper (Fig.  The Jung’s group presented a vacuum-pressure aided blood extraction system with Ni hollow MN for a colorimetric glucose sensor . They coper Fig. b, which per Fig. c. In terAn alternative method to the collection of ISF by using an MN array is to employ swelling materials for the body for the MN. It has been demonstrated that hydrogel MNs are capable to extract ISF by swelling, which results in a fluidic conduit –97, as wISF is absorbed in the dry hydrogel MNs by diffusion, after their penetration into the skin, which has to be followed by the process of separation of the sampled ISF from the MN array, by using centrifugation, either with or without immersion into a solvent. Although the swelling MNs intrinsically need the additional process to remove the analytes, the main fabrication process by mold casting is relatively simple and they have the ability to collect ISF efficiently due to the relatively large capacity of the sampling volume. This approach is also applied to the measurement of glucose levels , 97.Although the porous MNs were widely considered as drug delivery devices , 102–106The first successful study of a porous MN array for ISF collection and a fluidic connection to a external measurement system was reported by the Nishizawa’s group , 110. AsConsidering the research history of the micro-sized glucose sensors, MNs functionalized for bio-sensing systems are comparatively recent. Hence, there are still challenges to be faced in the development of MN-based bio-sensing devices, especially for CGMS.As discussed above, the MN-based electrodes, especially MNs coated with conductive layers, have been successfully indicated to monitor the blood glucose level continuously. This is because the MN-based electrodes are in direct contact with a sufficient amount of fluids beneath the skin for a reliable measurement of glucose, in a similar manner to conventional implantable sensors. However, from the perspective of the application of CGMS devices, the MN-based electrodes have certain drawbacks. One is a common problem of implantable sensors, that is, the loss of the sensitivity owing to surface fouling and enzyme inactivation induced by proteins and immune response –115. ThiOn the other hand, the use of MNs as biological fluid collectors have an advantage over their use as sensing probes because of the developed technology of ex vivo analysis of extracted biological samples. In particular, pTAS devices meet the purpose of the MN-based fluid collector by achieving a small amount of direct flow of ISF or blood from inside the skin to measurement systems. From this perspective, the hydrogel-forming swellable MNs have a disadvantage of the requirement of an additional step to separate the absorbed ISF from the MN array. As microfluidic CGMS devices composed of microchannels have already been proposed using infrared light , viscosiIn this paper, the applicabilities of functionalized MNs to CGMS were discussed. The MN-based in vivo sensors have been applied to diagnostic systems even in a clinical experiment level, by being functionalized as electrodes, especially for glucose measurement. However, the MNs as sensing probes have been facing the challenges similar to those of the implantable sensors, such as the impaired sensitivity and the limited measurement methodology. On the other hand, the MN-based trans-dermal fluidic channels can be highlighted with the potential to be combined with recently developed pTAS technologies. Therefore, the use of MNs as biological fluid collectors should be investigated for integration with the existing ex vivo diagnostic systems and for a more efficient sampling mechanism of ISF. The development of functionalized MNs in those ways will contribute to the realization of commercially available CGMS devices in a minimally invasive manner instead of conventional glucose monitoring devices with pain.


CTNNB1, AXIN, or APC, have been observed in the endometrioid and mucinous subtypes of EOC. In addition, upregulation of the ligands, abnormal activation of the receptors or intracellular mediators, disruption of the β-catenin destruction complex, inhibition of the association of β-catenin/E-cadherin on the cell membrane, and aberrant promotion of the β-catenin/TCF transcriptional activity, have all been reported in EOC, especially in the high grade serous subtype. Furthermore, several non-coding RNAs have been shown to regulate EOC development, in part, through the modulation of Wnt/β-catenin signalling. The Wnt/β-catenin pathway has been reported to promote cancer stem cell self-renewal, metastasis, and chemoresistance in all subtypes of EOC. Emerging evidence also suggests that the pathway induces ovarian tumor angiogenesis and immune evasion. Taken together, these studies demonstrate that the Wnt/β-catenin pathway plays critical roles in EOC development and is a strong candidate for the development of targeted therapies.Epithelial ovarian cancer (EOC) is the deadliest female malignancy. The Wnt/β-catenin pathway plays critical roles in regulating embryonic development and physiological processes. This pathway is tightly regulated to ensure its proper activity. In the absence of Wnt ligands, β-catenin is degraded by a destruction complex. When the pathway is stimulated by a Wnt ligand, β-catenin dissociates from the destruction complex and translocates into the nucleus where it interacts with TCF/LEF transcription factors to regulate target gene expression. Aberrant activation of this pathway, which leads to the hyperactivity of β-catenin, has been reported in ovarian cancer. Specifically, mutations of  Ovarian cancer is the most lethal gynecological malignancy and is ranked as the fifth leading cause of cancer deaths in females . It is eThe Wnt/β-catenin pathway regulates cell proliferation, polarity, survival, and stem cell fate in embryonic and adult tissue homeostasis . The patβ-catenin is the key mediator of the canonical Wnt pathway . In the β-catenin signalling is activated in the presence of Wnt ligands. On the cell surface, the binding of a Wnt ligand induces the heterogeneous dimerization of Frizzled (FZD) and LRP5/6 receptors, leading to their conformation change . DisheveCTNNB1 [CTNNB1 accounted for up to 54% of the EC cases [CTNNB1, the mutation was always found within the amino-terminal domain [The most common genetic alteration in the Wnt/β-catenin pathway involved in EOC is in the β-catenin gene, CTNNB1 . MutatioCTNNB1 . This isEC cases . In ECs l domain . Phosphol domain .Mutations in several components of the destruction complex, such as AXIN, GSK3β and APC, have been reported in EOC. Since these proteins are important for the degradation of β-catenin, genetic alterations that render them less effective or non-functional are likely candidates for driving hyperactive β-catenin signalling and, as a result, oncogenesis.CTNNB1, mutations in the genes encoding AXIN and APC proteins  have also been reported in EOC [AXIN1 has been found in one case of EC tumor, while a frameshift mutation in AXIN2 resulting in truncation has been found in another EC tumor [Although much less common than mutations in d in EOC , 32. AXIEC tumor . FunctioEC tumor .APC mutations in EOC has been controversial. For instance, it was once believed that the I1307K missense mutation in the APC gene conferred a modest increase in the risk of hereditary and sporadic breast/ovarian cancer development through its association with BRCA1/2 mutations. Later analysis, however, concluded that, although there exists a high prevalence of I1307K mutation amongst BRCA1/2 carriers, the I1307K allele confers no additional risk for cancer development [APC gene were identified in an MC tumor [APC mutations and the frequency at which these mutations occur in EOC.Genetic alterations in APC, while frequently detected in colon cancers, are rarely found in EOC , 33. As elopment . Two misMC tumor . While tCTNNB1 and components of the β-catenin destruction complex are rare or restricted to only the EC and MC subtypes, higher β-catenin activity is often observed in EOC, especially in HGSC. The mechanisms underlying the hyperactivation of the Wnt/β-catenin pathway in EOC are not entirely clear. However, many studies have reported the abnormal expression or activation of the components and regulators of this pathway. It is therefore highly possible that aberrant activities of these regulators contribute to the hyperactivation of Wnt/β-catenin in EOC, as summarized in Fig. Although mutations in in situ hybridization confirmed that this up-regulation occurred more frequently in serous than in EC, MC, and CCC tumors [In vitro functional analysis showed that downregulation of Wnt-7A reduced ovarian cell proliferation, adhesion, invasion and expression of β-catenin target genes, suggesting an important role in the activation of Wnt/β-catenin signalling and EOC development [Several Wnt ligands have been reported to be upregulated and associated with poor prognosis in EOC patients. For example, using immunohistochemistry, Wnt-5A expression was found to be strongly upregulated in EOC tumors when compared with benign epithelial neoplasia and normal ovarian samples and was negatively correlated with patient survival . This stC tumors . OverexpC tumors . In vitrelopment . The sigRSPO1) belongs to the secreted R-spondin protein family, which bind to the LGR4, LGR5 or LGR6 receptors [RSPO1 as an EOC susceptibility locus [RSPO1, RSPO2, LGR5 and LGR6 expression, although at low frequency, has also been observed [A recent study revealed that abnormal expression of R-spondin 1 also contributes to the dysregulation of the Wnt/β-catenin signalling pathway at the receptor level . R-spondeceptors . This bieceptors , therebyeceptors . A genomty locus . RSPO1 wty locus . In HSGCobserved . Moreoveobserved . Furtherobserved . Similarobserved . These fWnt/β-catenin signalling is tightly controlled by several negative regulators, some of which inhibit activation of Wnt signalling by competing with Wnt ligands for their receptors. Abnormal levels of these regulatory proteins have been associated with the dysregulation of Wnt/β-catenin activity and EOC development. For example, Dickkopf (DKK) inhibits Wnt signalling by binding to LRP5/6 and disrupting the dimerization of FZD and LRP5/6 . InteresDownregulation of DKK2 in EOC tumors is believed to involve epigenetic silencing. Compared to benign tumors and normal ovarian tissues, DKK2 in EOC tumors was more commonly methylated and its methylation was increased in higher grades and stages of EOC . Similaret al. revealed that there was a direct interaction between CCNG2 and DACT1 and that overexpression of DACT1 decreased DVL2 and β-catenin levels in gastric cancer cells [Cyclin G2 (CCNG2) is an unconventional cyclin which has been shown to inhibit cell proliferation, migration and invasion in EOC cells , 53. CCNer cells . While uer cells . Remarkaer cells . Recentler cells . Thus, dAnother cell cycle regulator, cyclin Y (CCNY), also regulates β-catenin signalling. CCNY has been found to be upregulated in EOC tissues and its expression to be positively correlated with the clinicopathological stage . In addiAPC in EOC tumors when compared to benign ovarian tumors or normal ovarian tissue samples [Decreases in the expression of certain components of the destruction complex are frequently observed in EOC . For exa samples –64. Howein vitro and tumor growth in nude mice, as well as aerobic glycolysis. Further studies confirmed that TNKS1 exerts these effects by promoting Wnt/β-catenin signalling [Tankyrases (TNKS), which belong to the poly (ADP-ribose) polymerase (PARP) family, are positive regulators of Wnt/β-catenin signalling , 66. TNKgnalling .Inhibition of GSK3β has also been observed in EOC , 69. Iniβ-catenin is a dynamic protein that can function as a component of adherens junctions or as a transcription factor depending on its subcellular localization. At the adherens junctions, β-catenin interacts with the cytoplasmic tail of E-cadherin and links E-cadherin to actin filaments through its interaction with α-catenin to maintain the dynamics of the cytoskeleton , 76. DisDuring EOC development, the membrane-associated β-catenin is dysregulated. Tissue transglutaminase 2 (TG2) has been shown to promote the dissociation of E-cadherin and β-catenin in EOC cells. TG2 was found to be overexpressed in EOC tumors and positively correlated with β-catenin levels in ovarian cancer cell lines , 80. TG2Several proteins are believed to modulate β-catenin activity within the nucleus. SOX7, a member of the Sox transcription factor family, was demonstrated to suppress Wnt signalling in ovarian cancer cells harboring either wildtype or mutant β-catenin . This waMultiple proteins have been reported to form a complex with β-catenin and TCF/LEF and increase the stability of the transcriptional complex. Pygopus (PYGO) binds β-catenin directly in the nucleus and assists in transcription of its target genes . PYGO2 hFOXM1, a member of forkhead transcription factors, plays important roles in EOC development in part by regulating β-catenin signalling. High FOXM1 levels were found to be correlated with EOC tumor grade and stage, and to predict poor prognosis and chemoresistance –91. InteNon-coding RNAs are RNA transcripts that do not encode proteins –96. HoweMultiple studies have shown the involvement of non-coding RNAs in EOC progression through the modulation of the Wnt/β-catenin pathway  . Like non of EOC , 143. Thn of EOC –149.Accumulating evidence points toward the Wnt/β-catenin pathway in playing an important role in the acquisition of stem-like properties in ovarian cancer cells , 150. AmPanax quinquefolius and notoginseng, and its metabolite, compound K, suppressed CSC self-renewal and inhibited β-catenin activity [Several studies have found that modulation of β-catenin activity altered the CSC-like properties. For example, miR-1207 suppressed SFRP1, AXIN2, and ICAT, three important negative regulators of the Wnt/β-catenin signalling pathway, to activate β-catenin signalling and promote the expression of CSC markers . Likewisactivity . Togethe+/CD44+ ovarian CSCs exhibited higher levels of resistance to paclitaxel and carboplatin [Recent studies encompassing ovarian CSCs and their involvement in EOC tumorigenesis reveal the association of CSCs and chemoresistance , 157. Thboplatin . Since Cboplatin  or inhibboplatin , 67, 91 in vitro and rendered the cells more susceptible to chemotherapeutic reagents, including cisplatin and paclitaxel [One of the mechanisms for chemoresistance in CSCs is the deregulation of membrane transporters, such as an ATP-binding cassette (ABC) transporter, ABCG2 , 159. Thclitaxel . hCRT1 iclitaxel . In cispclitaxel . It has clitaxel , 93. In clitaxel . Thus, iin vitro and in vivo [. These results and studies discussed above support the potential of Wnt/β-catenin inhibitors for the management of EOC patients with drug resistance.In addition to promoting resistance to conventional chemotherapies, a recent study provided evidence to support the activation of the Wnt/β-catenin signalling in inducing resistance to a PARP inhibitor, olaparib . Activat in vivo . These rEpithelial to mesenchymal transition (EMT) is a cellular process in which epithelial cells lose cell-cell adhesion and acquire mesenchymal characteristics, including migration and invasion . The attRecent evidence indicates that the activity and expression levels of E-cadherin and β-catenin are critical in the initiation of EMT in ovarian cancer cells . Loss ofin vitro [Additionally, Wnt/β-catenin signalling is involved in the remodeling of the extracellular tumor matrix in EOC, which is suggested to be mediated by the activities of matrix metallopeptidases (MMP). MMPs are proteolytic enzymes that act on diverse extracellular matrix (ECM) components such as fibronectin, gelatins, collagens, and laminins . MMP-2, in vitro . Furtherin vitro . Silenciin vitro .et al. (2018) examined the role of soluble E-cadherin in EOC and revealed that it interacted with VE-cadherin to induce angiogenesis [in vivo mouse models, is required to confirm the role of β-catenin in ovarian tumor angiogenesis.Tumor angiogenesis, wherein tumors promote blood vessel formation to provide themselves with nutrients and oxygen, is one of the hallmarks of cancer . Multiplet al. 20 examinedet al. 20 examined+ cells, which expressed IDO and IL-10, CD4+ T cells showed inhibition in responsiveness to antigen stimulation, suggesting IDO and IL-10 might be involved in the regulation of the immune response in EOC [Ovarian cancer has been reported to evade the immune system using multiple mechanisms, including the recruitment of regulatory T cells (Treg) and the promotion of T cell apoptosis via PD-L1 , 180. The in EOC . The same in EOC . HoweverCTNNB1, APC, and AXINs are restricted to the EC and MC subtypes of the EOC, hyperactivation of β-catenin is commonly observed in HGSC tumors and involves diverse mechanisms. This could be due to overexpression of ligands and receptors, underexpression of inhibitors of the Wnt/β-catenin pathway, and altered expression of proteins that regulate β-catenin/E-cadherin interaction on the membrane or β-catenin/TCF transcriptional activity, as summarized in Fig. Aberrant Wnt/β-catenin signalling has been widely linked to cancer development. Increasing evidence indicates that this pathway is hyperactivated in EOC and plays important roles in driving EOC development. Although mutations of in vitro. Therefore, it is important to confirm key findings in primary tumor cells collected from patients. Furthermore, EOC is composed of different histologic subtypes, each with distinct molecular features, mutational profiles, and even cellular origins [Compared to other types of cancer, especially colon cancer, fewer studies have been done on EOC to investigate the roles and mechanisms of the Wnt/β-catenin signalling pathway in the process of tumorigenesis. Nevertheless, evidence accumulated to date strongly supports a critical role for this pathway in promoting several key aspects of EOC development, from promoting CSC self-renewal, EMT and metastasis, drug resistance, and tumor angiogenesis, to suppressing tumor immunity. However, many of these studies were carried out using established cell lines and some of them were only conducted  origins . While tThe Wnt/β-catenin pathway is recognized as an important target for cancer therapy and many studies have been done to investigate the potential therapeutic effects of antibodies and small molecules that target this pathway and some of them are currently in clinical trials –188. How


The epigenetic factor protein arginine methyltransferase 5 (PRMT5) has been reported to play vital roles in a wide range of cellular processes, such as gene transcription, genomic organization, differentiation and cell cycle control. However, its role in pancreatic cancer remains unclear. Our study aimed to investigate the roles of PRMT5 in pancreatic cancer prognosis and progression and to explore the underlying molecular mechanism.18F-FDG (fluorodeoxyglucose) uptake by PET/CT (positron emission tomography/computed tomography) imaging were used to verify the role of PRMT5 in aerobic glycolysis, which sustains cell proliferation. The regulatory effect of PRMT5 on cMyc, a master regulator of oncogenesis and aerobic glycolysis, was explored by quantitative PCR and protein stability measurements.Real-time PCR, immunohistochemistry and analysis of a dataset from The Cancer Genome Atlas (TCGA) were performed to study the expression of PRMT5 at the mRNA and protein levels in pancreatic cancer. Cell proliferation assays, including cell viability, colony formation ability and subcutaneous mouse model assays, were utilized to confirm the role of PRMT5 in cell proliferation and tumorigenesis. A Seahorse extracellular flux analyzer, a glucose uptake kit, a lactate level measurement kit and the measurement of PRMT5 expression was significantly upregulated in pancreatic cancer tissues compared with that in adjacent normal tissues. Clinically, elevated expression of PRMT5 was positively correlated with worse overall survival in pancreatic cancer patients. Silencing PRMT5 expression inhibited the proliferation of pancreatic cancer cells both in vitro and in vivo. Moreover, PRMT5 regulated aerobic glycolysis in vitro in cell lines, in vivo in pancreatic cancer patients and in a xenograft mouse model used to measure 18F-FDG uptake. We found that mechanistically, PRMT5 posttranslationally regulated cMyc stability via F-box/WD repeat-containing protein 7 (FBW7), an E3 ubiquitin ligase that controls cMyc degradation. Moreover, PRMT5 epigenetically regulated the expression of FBW7 in pancreatic cancer cells.The present study demonstrated that PRMT5 epigenetically silenced the expression of the tumor suppressor FBW7, leading to increased cMyc levels and the subsequent enhancement of the proliferation of and aerobic glycolysis in pancreatic cancer cells. The PRMT5/FBW7/cMyc axis could be a potential therapeutic target for the treatment of pancreatic cancer.The online version of this article (10.1186/s12964-019-0344-4) contains supplementary material, which is available to authorized users. Pancreatic cancer is a devastating disease with an extremely high mortality rate. Although significant progress has been made in past decades in the diagnosis and treatment of pancreatic cancer, the overall 5-year survival rate of patients remains steady, at approximately 6% , 2. The Posttranslational methylation of histone lysine (K) or arginine (R) residues can regulate chromatin structures that lead to changes in gene expression, participate in the modulation of cellular processes and are often deregulated during cancer pathogenesis , 5. TherPRMT5 regulates the expression of a wide spectrum of target genes via modification of the chromatin structure or transcriptional machinery. Specifically, PRMT5 can catalyze the methylation of arginine 8 on histone H3 (H3R8) and arginine 3 on histone H4 (H4R3), resulting in the silencing of cell cycle genes such as cyclin E1 (CCNE1) . In addiIn the present study, our results demonstrated that PRMT5 was an unfavorable prognostic factor for pancreatic cancer. The results of in vitro cell line studies and in vivo subcutaneous mouse model experiments demonstrated that PRMT5 could regulate the tumorigenicity of pancreatic cancer cells. In-depth mechanistic explorations indicated that PRMT5 could regulate aerobic glycolysis via the regulation of cMyc stability by epigenetically silencing the expression of FBW7, a ubiquitin ligase and tumor suppressor with decreased expression in cancer cells , 27. ColThe human pancreatic cancer cell lines PANC-1, MIA PaCa-2, Capan-1, BxPC-3 and SW1990 were obtained from the American Type Culture Collection , and the cell lines were cultured according to standard protocols provided by ATCC. The human pancreatic ductal epithelial cell line HPDE was kindly provided by Professor Min Li at Ohio State University and routinely cultured in keratinocyte serum-free (KSF) medium supplemented with epidermal growth factor and bovine pituitary extract .Cells were washed twice with ice-cold PBS, harvested and lysed for 10 min in RIPA buffer  supplemented with protease and phosphatase inhibitors. For complete lysis, the lysates were sonicated and then centrifuged at 12000 rpm for 20 min at 4 °C. The concentrations of the lysates were determined by a BCA protein assay reagent kit . A total of 20 μg of total protein lysate was subjected to electrophoresis on denaturing 10% SDS-polyacrylamide gels and was then transferred to a membrane for subsequent blotting with specific antibodies. Antibodies against PRMT5, HIF1α and cMyc were purchased from Abcam . Antibodies against FBW7 were obtained from Bethyl Laboratories .Total RNA was extracted by using Invitrogen TRIzol reagent. To obtain cDNA, a TaKaRa PrimeScript RT kit  was used. The expression status of the designated genes was determined by real-time PCR using an ABI 7900HT Real-Time PCR system . To examine the mRNA expression status of PRMT5 in pancreatic cancer patients, 30 paired samples from patients diagnosed with PDAC were used. Total RNA was extracted from samples stored in RNAlater. β-Actin was used as the loading control. All reactions were performed in triplicate. The primer sequences are listed in Additional file To silence PRMT5 expression, the pLKO.1 TRC cloning vector  was used. The 21 bp targets against PRMT5 were GGCTCAAGCCACCAATCTATG and CCCATCCTCTTCCCTATTAAG, respectively. Scrambled shRNA or the Scr vector (Addgene plasmid 1864) was used as a knockdown control vector. The pCDH-CMV-MCS-EF1-Puro vector  was used to overexpress PRMT5, FBW7, and the corresponding mutants; empty vector (EV) was used as the control.To determine the roles of PRMT5 in pancreatic cancer cell proliferation, we performed in vitro cell viability assays by using a Cell Counting Kit-8  and colony formation assays according to previous reports .6 PRMT5-silenced or the corresponding control SW1990 cells were suspended in 100 μl of phosphate-buffered saline and injected subcutaneously into the back of the mice. The tumor size was measured weekly with calipers beginning at the formation of palpable tumors. The tumor volume was calculated by the following formula: length × width2 × 0.52. Six weeks after implantation, the mice were euthanized, and the tumors were surgically dissected. Samples were then processed for histopathological examination. All animal experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals and were approved by the IACUC of Fudan University.BALB/c-nu mice  were housed in sterile filter-capped cages. A total of 4 × 10To confirm the role of PRMT5 in aerobic glycolysis, we performed glycolysis measurements using a Seahorse Extracellular Flux Analyzer, an Abcam Glucose Uptake Assay Kit and a Biovision Lactate Colorimetric Assay Kit according to the protocols supplied by the manufacturers. In vivo glucose utilization by subcutaneous tumors was measured by using microPET/CT imaging to assess 18F-FDG uptake in xenograft tumors, according to our previous reports. Briefly, each tumor-bearing mouse was injected with 11.1 MBq (300 μCi) of 18F-FDG via the tail vein. Scanning started 1 h after injection. Animals were anesthetized with isoflurane during the scanning period. The images were reconstructed using a three-dimensional ordered subset expectation maximization (OSEM3D)/maximum a posteriori algorithm. Inveon Research Workplace was used to obtain the percentage injected dose per gram (%ID/g) and the SUVs . The SUVmax was calculated .18F-FDG was automatically made in a cyclotron (Siemens CTI RDS Eclipse ST) using an Explora FDG4 module. Patients were fasted for more than 6 h. Scanning started 1 h after intravenous injection of the tracer (7.4 MBq/kg). Images were acquired on a Siemens biograph 16HR PET/CT scanner with a transaxial intrinsic spatial resolution of 4.1 mm. CT scanning was initiated from the proximal thighs to the head at 120 kV and 80–250 mA with a pitch of 3.6 and a rotation time of 0.5 s. Image interpretation was carried out on a multimodality computer platform . Metabolic activity was quantified using the SUVs normalized to the body weights of the patients, and the SUVmax for each lesion was calculated [To assess the correlation between PRMT5 and glucose uptake in pancreatic cancer patients, we examined the SUVmax in pancreatic cancer patients via PET/CT imaging, a technique that measures glucose uptake via glycolysis by assessing 18F-FDG uptake. The SUVmax was obtained and calculated according to our previous reports. Briefly, lculated .The clinical pancreatic tumor samples used in the manuscript were obtained from patients diagnosed with pancreatic cancer at Fudan University Shanghai Cancer Center (FDUSCC), with approval from the Institutional Research Ethics Committee. Immunohistochemical staining of designated factors was performed according to our previous reports . ParatumTo confirm the impact of PRMT5 on FBW7 promoter activity, a dual-luciferase assay was performed. In brief, the promoter region of FBW7 covering the region from − 2000 to 200 base pairs was amplified from pancreatic cancer cells and ligated into the pGL3-Basic vector . PRMT5, pGL3-FBW7 and the control Renilla luciferase vector pRL-TK were cotransfected into cells, and luciferase activity was measured by using a Promega Dual Luciferase Reporter Assay System.ChIP assays were performed by using a Millipore EZ ChIP Kit. An antibody against PRMT5  was used for ChIP. ChIP-grade anti-histone H3 (acetyl K9), anti-histone H3 (tri methyl K9) and anti-histone 4 (symmetric di methyl R3) antibodies were purchased from Abcam. The primer sequences that covered the FBW7 promoter region with hypermethylation were F: 5′-CCCGGGAGAAGTGGCCCTGGACG-3′ and R: 5′-TTCGGACTGAAGCGGCAGCTGCGGA-3’ . A quanthttps://genome-cancer.ucsc.edu/). In total, 177 primary pancreatic cancer samples from patients with detailed expression data were selected from the updated TCGA database according to the parameters mentioned.TCGA-PAAD RNA expression data (Level 3) for pancreatic cancer patients obtained by RNA-seq by expectation-maximization was downloaded from the Cancer Genomics Brower of the University of California, Santa Cruz (UCSC)  using independent paired t-tests (two-tailed) or one-way analysis of variance (ANOVA). Differences were considered significant at *, The importance of PRMT5 expression in pancreatic cancer has seldom been discussed. First, by using quantitative real-time PCR and a paired t-test statistical analysis, we demonstrated that PRMT5 expression was higher in pancreatic adenocarcinoma (PDAC) tumor samples than in adjacent samples Fig. a. Next, To confirm the roles of PRMT5 in tumorigenicity, we performed in vitro proliferation assays. First, we examined the expression status of PRMT5 in pancreatic cancer cell lines, including HPDE, Capan-1, PANC-1, BxPC-3, MIA PaCa-2 and SW1990. The results of western blots with the anti-PRMT5 antibody demonstrated that the PRMT5 level was higher in MIA PaCa-2 and SW1990 cells, while HPDE and Capan-1 cells displayed lower protein levels of PRMT5


To determine the role of the dishevelled binding antagonist of beta catenin 1 (DACT1) in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation (AF).The DACT1 expression and its associations with the degree of fibrosis and β-catenin in valvular disease patients were analyzed by immunohistochemistry and Masson’s staining. DACT1 was overexpressed in the atrial myocyte cell line (HL-1) and the cardiac cell line (H9C2) by adenoviral vectors. Alterations in the fibrous actin (F-actin) content and organization and the expression of β-catenin were detected by flow cytometry, immunofluorescence, and Western blotting. Additionally, the association of DACT1 with gap junctions connexin 43 (Cx43) was detected by immunohistochemistry, immunofluorescence, and Western blotting.P=0.037) and a high degree of fibrosis . A positive association was observed between DACT1 and β-catenin expression in clinical samples . Furthermore, overexpression of DACT1 in HL-1 and H9C2 cells induced an increase in β-catenin and subsequent partial colocalization of DACT1 and β-catenin. In addition, F-actin content and organization were enhanced. Interestingly, DACT1 was positively correlated with the Cx43 expression in clinical samples  and changed the Cx43 distribution in cardiac cell lines.Decreased cytoplasmic DACT1 expression in the myocardium was associated with AF (DACT1 proved to be a novel AF-related gene by regulating Cx43 via cytoskeletal organization induced by β-catenin accumulation in cardiomyocytes. DACT1 could thus serve as a potential therapeutic marker for AF. The cytoskeleton plays an important role in this process, because of its involvement in the channel opening mediated by integrin in the process of the force transmitting force indirectly to stretch-activated channels (SACs), as well as the regulation of L-type Ca2+ current  and signaling pathway transduction. Moreover, the force transmission can be directly or indirectly mediated by the focal proteins on the cytoskeleton. Acute mechanical changes produce electrophysiological alterations and arrhythmias, mediated by both the cytoskeleton and the extracellular matrix-6. Once AF is induced, rapidly and inhomogeneously contracting and interacting atrial segments would tend to perpetuate electrophysiological dispersion, which could facilitate the geometry (fibrosis and scarring of atrium) formation. Thus, the cytoskeleton could be an essential systematic regulator and a potential therapy marker for AF.Atrial fibrillation (AF) is a ubiquitous arrhythmia and causes considerable morbidity and mortality; however, its mechanism remains unclear. AF is known to be caused by significant atrial electrical and structural remodeling. β-catenin can affect both the cellular microtubule arrays by interacting with the complex of EB1/P150/dynein/dynactin and P120-catenin and affect the organization of the cellular actin filament by interacting with actin-bundling protein,10. In cardiac cells, β-catenin directly binds to the C-terminal region of cadherin, whereas α-catenins link the cadherin/catenin complex to the actin cytoskeleton. Thus, β-catenin is essential for the maintenance of cardiac cellular function. Abnormal β-catenin in vivo could lead to hypertrophic cardiomyopathy, heart failure, and arrhythmias,13. However, the mechanism of cytoskeletal arrangement via β-catenin involvement in AF remains unclear.β-catenin is not just an important transcriptional effector of the canonical Wnt signaling pathway in the nucleus, but a crucial regulator of the cytoskeleton and signal transduction outside the nucleus as well. Yuan et al. suggested that DACT1 induced the β-catenin accumulation in the cytoplasm by inhibiting the glycogen synthase kinase beta (GSK3-β) activity and interacting directly with β-catenin. The actin cytoskeleton, which may be regulated by β-catenin, serves as a link to the adherens junctions, gap junctions, and desmosomes, which constitute the intercalated disc. The remodeling of gap junctions, which are assembled from connexins, has been acknowledged to be an important contributor to AF. Thus, we hypothesized that a new mechanism mediated by DACT1 might exist by which the stability of gap junctions is regulated in AF. However, little is known about this mechanism.A previous study showed that the dishevelled binding antagonist of beta catenin 1 (DACT1), a novel bidirectional regulator of β-catenin, was associated with cytoplasmic β-catenin accumulation, suggesting its potential role in cytoskeletal arrangementIn the present study, we demonstrated that DACT1 is involved in AF. Then, we demonstrated that DACT1 was involved in AF by regulating the actin cytoskeleton. Furthermore, we found out that connexin 43 (Cx43) remodeling might be the result of the effect of DACT1 on the cytoskeleton in AF.This study was approved by the Human Ethics Committee of the First Affiliated Hospital of Sun Yat-Sen University and complied with the principles governing the use of human tissues that are outlined in the Declaration of Helsinki. Informed consent was given by patients before participating in the study.Tissue samples from the right atrial (RA) appendage were obtained from 29 patients with valvular heart disease who had undergone valve replacement surgery. Ten patients, who constituted the sinus rhythm (SR) group, did not have a history of AF, and 19 patients, who constituted the AF group, had documented arrhythmias from which they had suffered for more than six months before undergoing surgery. These tissue samples were obtained during the surgeries, before the aortic cross-clamping, and were immediately fixed in 4% paraformaldehyde (PFA). The diagnosis of AF was made based on patient medical records and 12-lead electrocardiogram (ECG) findings. All subjects underwent ECG to verify the underlying SR and an echocardiogram finding to document the preserved valvular and myocardial function. Preoperative functional statuses were recorded in accordance with the New York Heart Association (NYHA) classification. Patients’ data are summarized in The RA appendages of normal hearts were obtained from autopsies (two cases) and provided by the Department of Forensic Pathology of Shantou University Medical College and the Department of Forensic Pathology of Jiaxing University Medical College. The ages of the both male patients were 19 and 23 years. The hearts obtained at autopsy were devoid of any abnormal findings and the causes of death were not heart related.All the samples were fixed in 4% PFA, embedded in paraffin, and stained with hematoxylin and eosin for routine histological examination. Immunohistochemical staining was performed on 4-µm-thick tissue sections. After deparaffinization and rehydration, all the sections were microwaved (10 min) in 0.01 mol/L sodium citrate buffer (pH 6.0) for antigen retrieval. To block endogenous peroxidase activity, we incubated the sections with 10% normal goat serum in phosphate-buffered saline (PBS) for 15 min at room temperature. Then, all the sections were incubated with a rabbit polyclonal antibody against DACT1 , β-catenin , or Cx43  overnight at 4°C. The slides were subsequently treated with a SuperPic Ture Polymer Detection Kit and Liquid DAB Substrate Kit  and counterstained with hematoxylin, dehydrated, and mounted.The sections were stained with Masson’s trichrome for fibrosis quantification. For Masson’s trichrome staining, the sections were dewaxed with xylol . All the slices were then washed in distilled water and placed in a hematoxylin solution for 3 min, after which a color change was induced with lithium carbonate. The slices were subsequently washed in pure water and colored with Ponceau red staining (in a 45 kW oven at 30°C for 20 sec). Next, the slices were placed in distilled water and phosphomolybdic acid for 1 min before being labeled with a green fluorescent marker and washed again with distilled water. The severity of fibrosis was then assessed in each of the sections upon their collection.Immunohistochemical expression was evaluated using the Image-Pro Plus 6.0 software . Briefly, at least three positive expression fields in a section of myocardial tissue were randomly selected, and then these positive regions were analyzed with the Image-Pro Plus 6.0 software to determine their integral optical density and area. The average of the optical density values, which represented the expression intensity in the section, was subsequently calculated.Fibrosis severity was evaluated using the Image-Pro Plus 6.0 software. At least three fields in a section of myocardial tissue were randomly selected after which the ratio of the fibrotic area to the total area of each selected field was calculated to assess fibrosis severity. The average ratio, which represented the severity of the fibrosis in the section of myocardial tissue, was subsequently determined.E. coli BJ5183 cells to obtain pAd5-D1V1-GFP and pAd5-D1V2-GFP, respectively. pAd5-D1V1-GFP and pAd5-D1V2-GFP were amplified in competent E. coli XLI-Blue cells and prepared with a Qiagen Plasmid Midi kit (QIAGEN), according to the manufacturer’s protocol. pAd5-D1V1-GFP and pAd5-D1V2-GFP were then linearized and transfected into HEK293 cells to rescue and propagate the respective recombinant adenoviruses. The expression levels of DACT1 were analyzed further by Western blotting analysis, and GFP-expressing cells were observed using a fluorescence microscope (Axio Observer Z1).Briefly, the complementary deoxyribonucleic acid (cDNA) of two transcript variant isoforms of DACT1 (named D1V1 and D1V2) were purchased from GeneCopoeia , and inserted into pGA1-2A-GFP through enzymatic digestion and ligation to obtain pGA1-D1V1-GFP and pGA1-D1V2-GFP. The autocleavage 2A linker between DACT1 and green fluorescent protein (GFP) facilitates the formation of separated DACT1 and GFP reporter. Subsequently, pGA1-D1V1-GFP and pGA1-D1V2-GFP were linearized by enzymatic digestion and subjected to homologous recombination with the linearized pAd5∆E1∆E3 backbone in competent 2 atmosphere at 37°C.The H9C2 and HL-1 cell lines were used in this study. The HL-1 cell line was obtained from EMD Millipore Corporation (Cat. SCC065). Cells were cultured using Claycomb medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/L streptomycin in flasks precoated with fibronectin and gelatin. The H9C2 cell line was cultured in Dulbecco's Modified Eagle Medium (DMEM)/high-glucose medium (HyClone) with 15% FBS and antibodies (100 U/mL penicillin and 100 mg/L streptomycin). All the cells were grown under a 5% COCells were plated 24 hours before transduction, incubated with fresh media containing the required multiplicity of infection (MOI) per cell of the virus, left for 18 hours, washed, and maintained until harvesting. Forty-eight hours after transduction, GFP-expressing cells were observed using a fluorescence microscope (Axio Observer Z1).. Proteins were isolated from cells with lysis buffer  that included a protease inhibitor cocktail . Proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes . Primary antibodies against DACT1 , β-catenin , or Cx43  were used, and antigen-antibody complexes were detected by a Western blotting luminol reagent (Santa Cruz Biotechnology). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech) or β-actin (Wuhan GoodBio Tech. CO. LTD) served as an internal reference, and at least two independent experiments were performed for each cell line. Image J software was used to analyze the mean light density of each band. The expression of target genes was normalized to that of GAPDH or β-actin.The technique used was described previously. The F-actin analysis was conducted via flow cytometry, and cells were fixed with 70% ethanol overnight at 4°C. Cell pellets were incubated in PBS containing 0.1% Triton-X 100 for 15 min at room temperature and then they were treated with phalloidin-coumarin  for 30 min. The F-actin content was analyzed by flow cytometry . FlowJo 7.6 software  was used to analyze the F-actin content in each group.The technique used was described previously. Cells were fixed with 4% PFA for 15 min, blocked, and permeabilized with 1% FBS and 0.2% Triton X-100 in PBS at room temperature for one hour. Then, the cells were incubated with primary antibody overnight at 4°C. Primary antibodies against DACT1 , β-catenin , or Cx43  were used at the indicated dilutions. After the cells were washed three times with PBS, secondary antibody  was diluted at 1:500 and applied to the cells for one hour at room temperature. For F-actin staining, the cells were incubated with 100 nmol/L Alexa Fluor® 647 Phalloidin (Thermo Fisher). Finally, the cells were incubated with 1µg/mL 4,6-diamidino-2-phenylindole (Sigma). Cells were analyzed by confocal fluorescence microscopy (Zeiss 780 NLO).The technique used was described previously2-tests. Continuous variables are presented as the mean ± SEM. Comparisons of continuous variables between groups were performed with Student’s t-test, and the correlations between DACT1 expression levels and β-catenin or Cx43 were assessed with the nonparametric Spearman’s rank correlation test. P<0.05 was considered to indicate statistical significance. All statistical analyses were performed with Statistical Package for the Social Sciences (SPSS) software, version 19.0.Correlations between the immunohistochemical results and patient clinical variables were analyzed by χP=0.037, vs. strong, P=0.028; weak vs. very strong, P=0.029)   to 1G. T; thus, we generated an adenovirus with two isoforms of DACT1 (named V1 or V2 in this study) for further investigation, and the overexpression efficiency was detected   . Moreoveo=0.408) . No DACTThe concentration and distribution of F-actin, an essential element of the cytoskeleton, were then detected. Phalloidin-coumarin staining indicated that DACT1 overexpression increased the F-actin concentration . Further, we hypothesized that the Cx43 might be involved in the regulation of the cytoskeleton by DACT1 in myocardial cells. High Cx43 expression was associated with low fibrosis degree, right ventricular (RV), and RA values (Given that cytoskeletal reorganization could lead to an alteration of expression and cellular distribution of Cx43 and gap junction remodelingA values , suggestA values ; howeverA values , suggestA values . We spec. Despite the recent significant advances in the understanding of the mechanisms associated with AF, complexities in the etiology of atrial electrical dysfunction have prevented definitive elucidation.AF, the most common cardiac arrhythmia in clinical practice, is a major cause of embolic stroke and human diseases by affecting β-catenin. It was proposed that DACT1 was very important in human diseases at least because of the contribution for determining the role of β-catenin, a structural protein and/or a signal transduction protein. Our investigation in the present study confirmed this hypothesis. In the myocardium of the patients, β-catenin was not expressed in the nucleus, but was present in the cytoplasm and/or membrane. Furthermore, its expression was positively correlated with DACT1 cytoplasmic expression, suggesting that DACT1 may facilitate the extranuclear accumulation of β-catenin, which might serve as a structural protein in the myocardial cells of the valvular heart disease patients. Further analysis of the myocardial structure confirmed this speculation. DACT1 induced F-actin filament accumulation in the myocardial cells. Actin filaments reorganization can lead to alteration of the gap junction protein Cx43. Thus, unsurprisingly, we found a correlation between the expression of DACT1 and Cx43 in clinical samples and found that DACT1 altered the cellular distribution of Cx43 in the cardiac cell line. Cx43 is a very important susceptibility gene in AF, and the reduced number and organization of Cx43-containing gap junction plaques likely play a fundamental role in the increased incidence of arrhythmias and degree of fibrosis,26. We also found the structural and fibrotic relevance of Cx43 (DACT1, which was first found as a Dvl-interacting protein, plays a crucial role in the normal vertebrate development of Cx43 , and theIn summary, we demonstrated for the first time that a lack of DACT1 was associated with AF and the degree of fibrosis in valvular heart disease. Specifically, DACT1 regulated the gap junction protein Cx43 by accelerating the cytoskeleton rearrangement via accumulation of β-catenin in the myocardial cells, indicating that new mechanisms mediated by DACT1 exist in AF. DACT1 is a novel potential therapeutic target for AF.