However, the role of the homolog Pygo1 in human lung cancer remains unclear. In the current study, we demonstrated an association of increased Pygo1 expression with consistent high nuclear β-catenin signals across pathological tissue samples of early-stage human NSCLC. Overexpression of Pygo1 in lung cancer cells resulted in enhanced G1/S cell phase transformation, reduced apoptosis, and increased cell proliferation. These changes were accompanied by the downregulation of cell cycle-related proteins, such as RB, p16, p53, and p27Kip1, and increased expression of CyclinE1. Migration, wound healing, and colony formation assays revealed that Pygo1 overexpression enhanced the invasion and migration of lung cancer cells, increased the formation of clones, and suppressed E-cadherin expression. In addition, overexpression of Pygo1 in lung cancer cells led to an increase of β-catenin/TCF4 complex, as well as upregulated expression of target genes of β-catenin. In vivo experiments also revealed that Pygo1 overexpression promoted the tumorigenicity of a xenograft tumor model, while Wnt inhibition partially blocked the effect of Pygo1 overexpression. In conclusion, Pygo1 affects human NSCLC via the canonical Wnt/β-catenin pathway, which provides new clues for lung cancer pathology.Abnormal activation of the classical Wnt pathway has been reported in non-small-cell lung cancer (NSCLC) previously. Pygo family genes, the core regulators of Wnt/ Lung cancer is the main cause of cancer-related deaths worldwide , and theβ-catenin [β-catenin and further induces its ubiquitination and degradation to maintain a low β-catenin level in the cytoplasm. In the absence of nuclear β-catenin, histone deacetylases (HDACs) are recruited by inhibitor complexes containing T cell factor (TCF)/lymphoid enhancer-binding factor 1 (LEF) and transducing-like enhancer protein (TLE/Groucho) to prevent the expression of target genes [β-catenin protein enters the nucleus and forms a complex with TCF/LEF and B cell lymphoma-9 (Bcl-9) to regulate the expression of downstream target genes [In animals, the Wnt signaling pathway is considered highly important , which h-catenin , which f-catenin . The comet genes . In the et genes .β-catenin, are associated with colon cancer or colorectal adenomatous polyposis but rare in lung cancer [β-catenin in lung cancer.Abnormal activation of the classical Wnt signaling pathway has been identified in a variety of human cancers. For example, the tumorigenesis and prognosis of NSCLC are associated with the abnormal activation of Wnt, which is accompanied by the silencing of Wnt inhibitory factor-1 and the methylation of secreted Fzd-related proteins . Howeverg cancer , suggestDrosophila studies. It was reported that the nuclear accumulation of β-catenin required the interaction of Pygo and Lgs during the formation of a typical transcription complex [β-catenin/TCF, the N-terminal homology domain of Pygo activates the expression of target genes [β-catenin affects lung cancer development.Pygopus (Pygo) has been identified as a critical component of canonical Wnt signaling in genetic  complex . It has et genes , such aset genes . Pygo2 oet genes . Howeverhttp://kmplot.com/analysis/index.php?p=service&cancer=lung) was used to analyze the impact of Pygo1 on overall survival (OS) in lung cancer patients. The Gene Expression Omnibus database (GEO), Cancer Genome Atlas (TCGA), and European Genome-Phenome Archive (EGA) were reassessed. The resulting curves were plotted based on the Pygo1 expression value.To investigate the clinical relevance of Pygo1 in NSCLC, the Kaplan-Meier plotter  classification system. Both NSCLC tumor tissues and adjacent normal tissues were removed during the resection surgeries.μg/ml; Sigma-Aldrich, USA). Stable transfectants were incubated in conventional medium with the addition of G418 (500 μg/ml).A549 cells  were cultured in RPMI-1640 medium complemented with 10% fetal bovine serum  and 100 U/ml streptomycin/penicillin . Lipofectamine 2000 reagent (US Life Technologies) was used for the transient transfection of cells with a Tag2B-labeled Pygo1-encoding plasmid and corresponding empty plasmid vector, according to the manufacturer's instructions. Another group was transfected with Tag2B-labeled Pygo1-encoding plasmid and corresponding empty plasmid vector and both treated with Wnt inhibitor KYA1797K . Besides, the cells transfected with Tag2B-labeled Pygo1-encoding plasmid were transferred to a 6-well plate for selection with G418 . After centrifugation at 1000 × g for 5 min, the supernatant was discarded and the cells were fixed with 70% ice-cold ethanol overnight at 4°C prior to resuspension in 0.5 ml of PI/RNase Staining Buffer. After a 10 min incubation at room temperature in a dark room, the cell cycle phases and apoptosis were analyzed using a Gallios Flow Cytometer .A549 cells were harvested 48 h after transfection with the Pygo1 overexpression plasmid or empty vector control plasmid. More than 103 A549 cells were seeded in each well of a 96-well plate for 24 h, transfected with the indicated plasmid and/or inhibitor, and incubated for an additional 24, 48, or 72 h. 10 ml of CCK-8 reagent was added to each well 1 h before the endpoint of the incubation. Finally, the optical density at 450 nm (OD450) in each well was determined by a microplate reader.The cell proliferation rates were measured using Cell Counting Kit-8 . A total of 5 × 10μm pores . At 48 h after transfection, 3.0 × 105 A549 cells in serum-free medium were added to each Matrigel matrix-precoated  upper well insert. Then, 500 μl of medium containing 10% fetal bovine serum was added to each lower chamber. After a 48 h incubation, the noninvaded cells were removed from the upper surfaces of the Transwell membranes with a cotton swab, and the cells that had invaded the lower membrane surface were fixed in methanol, stained with 0.5% crystal violet, photographed, and counted. In addition, the chambers were removed, placed in 500 μl of 10% acetic acid, shaken slowly to decolorize the chambers, and subjected to a measurement of the absorbance (OD) value at 550 nm using an enzyme-standard instrument.An invasion assay was conducted using Transwell inserts with 8 5 cells per well. Horizontal lines were evenly drawn on the back of the orifice plate. Once the cells formed an adherent monolayer, a pipette tip and a ruler were used to draw a horizontal line perpendicular to the previous drawing. The cells were washed 3 times with sterile PBS to remove the detached cells, and the medium was replaced with serum-free Dulbecco's Modified Eagle's Medium. The scratches were imaged at baseline (0 h) and cultured at 37°C and 5% CO2 for 24 and 48 h. An image was collected at each time point.For the migration assay, the cells were digested with trypsin, counted, and seeded in a 6-well plate at a density of approximately 5 × 103 cells per dish and were incubated for 2 weeks. The formed colonies were fixed with 10% formalin, stained with Giemsa, and counted.After transfection and inhibitor treatment, cells were plated evenly in 60 mm culture dishes at a density of 1.2 × 10g and 4°C for 15 min. The supernatants were moved to fresh microcentrifuge tubes placed on ice. A nuclear protein extraction kit  was used for nuclear protein extraction. Equal amounts of protein lysates were loaded onto a SDS-PAGE gel, separated by electrophoresis, and electrotransferred to nitrocellulose membranes . After a 2 h incubation in 5% nonfat dried milk in TBST at room temperature to block nonspecific binding sites, the membranes were incubated overnight at 4°C with primary antibodies against human Pygo1 , c-Myc , survivin , E-cadherin , cleaved-caspase-3 , CyclinD1 , RB , p16 , p53 , β-catenin , p27Kip1 , CyclinE1 , GAPDH , and β-actin , respectively. The membranes were washed 3 times with TBST and incubated with a HRP-conjugated secondary antibody for 2 h at room temperature . After 3 washes with TBST, the membranes were finally incubated in Luminata Forte Western HRP Substrate  for 5 min and scanned using an Odyssey Infrared Imaging System . The labeled protein bands were quantified using ImageJ software , and the data were normalized to the expression levels of GAPDH proteins. The blots shown in the figures were representative of at least 3 experiments.The cells were harvested and washed with ice-cold PBS. After aspirating the supernatant, the cells were resuspended with ice-cold RIPA buffer containing protease and phosphatase inhibitor cocktails . The cell suspensions were continuously agitated for 30 min at 4°C and then centrifuged in a microcentrifuge at 12,000 × μl of protein A/G-Magnetic beads . The supernatants were immunoprecipitated with 5 μg of anti-β-catenin antibodies overnight at 4°C, followed by incubation with protein A/G-agarose 4 h at 4°C. Negative control was immunoprecipitated with rabbit IgG or mouse IgG only. Protein A/G-Magnetic bead-antigen-antibody complexes were collected by centrifugation at 12,000 rpm for 60 s at 4°C. The pellets were washed 5 times with 1 ml of IPH buffer  for 5 min each time at 4°C. Bound proteins were resolved by SDS-PAGE, followed by western blotting with anti-β-catenin or anti-TCF4  antibodies, respectively. The experiments were replicated ≥3 times.Protein solution was quantified using the Bradford method, and equal concentration was used from all samples. Cell extracts were first pretreated with 25 6 cells in a volume of 100 μl was s.c. injected into the flanks of male mice (3 mice/group). When the mean tumor size reached between 100 and 200 mm3, the mice were randomly divided into 2 groups . KYA1797K (in a suspension of 90% PBS and 10% Tween 80) was injected i.p. 5 d per week at a dose of 20 mg/kg. Tumor volume and body weight were measured every 4 d. Tumors were measured using Vernier calipers, and tumor volume was calculated according to the following formula: V = 0.5(L × W2), where L is length and W is width. 40 days after drug treatment, the mice were killed, and the tumors were excised, weighed, and fixed in 4% PFA or snap-frozen in liquid nitrogen for further analysis.The mouse experiments were conducted in the animal facility of the College of Life Sciences, Hunan Normal University , and approved by the Institutional Animal Care and Use Committee. Approximately 4-week-old male athymic Balb/c-nu/nu mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. . Mice were housed in a microventilation cage system (MVCS) with a computerized environmental control system (Threeshine Inc.). The temperature was maintained at 24°C with a relative humidity of 45-55%. The mice were provided a standard maintenance diet (Dae Han Bio Link). After acclimatization for 1 week, A549 cells stably transfected with control and Pygo1-encoding plasmids were trypsinized and resuspended in phosphate-buffered saline (pH 7.4). The cell suspensions were then mixed with Matrigel  at 4°C. The mixture containing 5 × 10t-test was used to compare the means of continuous variables between 2 groups. Statistical significance was indicated by ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.01. A p value of <0.05 was considered to indicate a significant difference.The data were presented as the means ± standard deviations (SD). GraphPad Prism 8.0  was used to conduct the statistical analysis. Student's To explore the clinical significance of Pygo1 in NSCLC, we analyzed the relationship between Pygo1 expression and overall survival (OS) using the Kaplan-Meier Plotter public database. A significant association of higher Pygo1 expression with reduced patient survival rates was observed . Tissue To determine the role of Pygo1 in the tumorigenesis of NSCLC, we next subjected A549 cells to transient transfection with a Pygo1 overexpression vector or empty Tag2B vector (control) Figures . The proin vitro CCK-8 assay demonstrated that Pygo1 overexpression caused a significant increase of cell proliferation , ZEB1, and ZEB2 in cancer cells, thereby reducing the level of E-cadherin to promote cell migration [β-catenin molecules, thus enabling an increase in the nuclear level of β-catenin and further promotion of cell migration.Our current observations also suggested that Wnt/ migrate , 32. Stuigration , 34. Theβ-catenin signaling, which seems to occur in human lung cancer patients. Consistent increased β-catenin nuclear signaling was observed in human Pygo1 highly expressed NSCLC tissues. Moreover, in vitro experimental data showed that overexpression of Pygo1 in lung cancer cells could promote β-catenin/TCF4 complex formation. The mechanism of these changes might have two reasons. One was that the high expression of Pygo1 enhances the affinity of β-catenin and TCF4 [β-catenin. In our data, overexpression of Pygo1 did upregulate β-catenin expression, which might be a positive feedback mechanism in the Wnt/β-catenin signaling pathway, because our data also showed that β-catenin expression was slightly downregulated after KYA1797K treatment. It appears to be a positive feedback mechanism in the Wnt/β-catenin signaling pathway, which is coincident with a recent publication [Interestingly, Pygo1 promotes cancer through Wnt/and TCF4 ; the othlication .β-catenin signals in the nucleus [β-catenin level in lung cancer patients, indicating that Pygo1 induced lung cancer through a β-catenin-dependent mechanism.In any case, it is understandable that the overexpression of Pygo1 in patient tissue cells leads to the accumulation of  nucleus . Our resWe noted that in our study overexpression of Pygo1 was accompanied by a decrease of P53 levels. Previous studies have shown that there is a regulatory relationship between P53 and the Wnt signaling pathway, but it is unclear whether the same situation exists in this study . The mec


We investigated the therapeutic effects of microRNA-139-5p in relation to osteoporosis of bone marrow-derived mesenchymal stem cell (BMSCs) and its underlying mechanisms. In this study we used a dexamethasone-induced in vivo model of osteoporosis and BMSCs were used for the in vitro model. Real-time quantitative polymerase chain reaction (RT-PCR) and gene chip were used to analyze the expression of microRNA-139-5p. In an osteoporosis rat model, the expression of microRNA-139-5p was increased, compared with normal group. Downregulation of microRNA-139-5p promotes cell proliferation and osteogenic differentiation in BMSCs. Especially, up-regulation of microRNA-139-5p reduced cell proliferation and osteogenic differentiation in BMSCs. Overexpression of miR-139-5p induced Wnt/β-catenin and down-regulated NOTCH1 signaling in BMSCs. Down-regulation of miR-139-5p suppressed Wnt/β-catenin and induced NOTCH1 signaling in BMSCs. The inhibition of NOTCH1 reduced the effects of anti-miR-139-5p on cell proliferation and osteogenic differentiation in BMSCs. Activation of Wnt/β-catenin also inhibited the effects of anti-miR-139-5p on cell proliferation and osteogenic differentiation in BMSCs. Taken together, our results suggested that the inhibition of microRNA-139-5p promotes osteogenic differentiation of BMSCs via targeting Wnt/β-catenin signaling pathway by NOTCH1. Bone tissue engineering (BTE) brings new opportunities for the repair of bone defects and bone reconstruction . Due to As a type of key seed cell in osteogenic differentiation as well as in bone development and reconstruction, stem cells are regulated by classic epigenetics. In addition, it is also being discovered through more and more studies that miRNA plays an important regulatory role in osteogenic differentiation , 5. As aThe Notch gene was first discovered in fruit fly, which was so named since its loss of some function led to the notch in the fruit fly wing margin. The Notch signaling pathway exists extensively in multiple vertebrate and invertebrate species, and participates in regulating the vital activities in a series of physiological and pathological processes, including cell fate determination, differentiation, proliferation, apoptosis, epithelial-mesenchymal transition (EMT), angiogenesis, migration and adhesion. As is shown in relevant research, the Notch signaling pathway plays different roles in the differentiation of different stem cells, and different Notches have different effects even in the differentiation of the same stem cell.The Wnt protein family is a group of secretory proteins that are involved in regulating numerous embryonic development processes . The claThe animal protocol was approved by the Committee on the Ethics of Animal Experiments of Second Affiliated Hospital of Zhejiang University. C57BL/6 mice  were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China) and were given free access to food and water. They were caged individually under 22-24°C and 55-60% humidity with a 12 h light cycle. The present study used a dexamethasone-induced model of osteoporosis, as described previously [g for 10 min, and re-suspended using red blood cell (RBC) lysis buffer  for 10 min. Cells were centrifugated at 300 ×g for 10 min, and were re-suspended using PBS. Cells were re-suspended in DMEM supplemented with 10% fetal bovine serum  in culture flasks at a density of 1 × 105 cells/ml. Adherent cells were cultured by changing the medium every 3 days and the cells were used for subsequent experiments following 3 passages.Male C57BL/6 mice  were sacrificed by cervical dislocation under anesthetization and disinfected with 75% alcohol for 3-5 min. The femurs and tibiae were carefully isolated and flushed with Dulbecco’s modified Eagle’s medium . Cells were centrifugated at 300 ×BMSCs were seeded into a 6-well plate and cultured overnight to reach 70% confluence. LV-miR-139-5p-overexpressing, si-NOTCH1 and LV-NC (negative control) were purchased from Hanbio . Subsequently, cells were transfected with different lentiviruses in the presence of ploybrene  for 24 h.3 cells/well for 48 h. MTT assay  was added into wells and cultured for 4 h at 37°C. Medium was replaced, and DMSO assay was added into wells and cultured for 20 min at 37°C. Absorbance was measured at 490 nm using a microplate reader .After lentivirus-mediated cell transfection for 24, h, cells were seeded in 96-well culture plates at a density of 0.5 × 10Total RNAs from cultured BMSCs were extracted using RNA Pure Rapid Extraction Kit . Total RNAs was amplified to complimentary DNA (cDNA) using a high-capacity cDNA reverse-transcription kit . RT-PCR was quantitatively determined using the SYBR Green Dye system (SYBR Premix Ex Taq (Tli RNase Plus) by an Applied Biosystems StepOnePlus real-time PCR System (USA). RT-PCR was as follows: at 95°C for 10 min, then 40 cycles consisting of 95°C for 15 sec, 60°C for 30 sec, and 72°C for 1 min, followed by 5 min incubation at 4°C.The 3'UTR seed region of Notch1 was synthesized and cloned into a pMIR-REPORT luciferase vector . The Notch1 construct or control construct was co-transfected into C BMSCs with miR-139 using Lipofectamine 2000 . After 48 h of transfection, luciferase activity was measured using the Dual Luciferase Reporter Assay system .BMSCs were lysed in RIPA buffer  supplemented with PMSF (Beyotime) and total proteins were quantitatively determined using a BCA Protein Assay Kit (Beyotime). Equal total proteins were separated by 8-10% SDS-PAGE and transferred onto PVDF membranes . Membranes were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore). Membranes were blocked with 5% non-fat milk in TBST for 1 h, and then incubated overnight at 4°C with primary antibody against: Wnt, β-catenin, NOTCH1 and GAPDH. After washing with TBST, the blots were incubated with HRP-conjugated rabbit anti-goat IgGs at 37°C for 1 h and then visualized using enhanced chemiluminescence reagents.Cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min. Next, cells were blocked for 30 min using 0.25% TrisX-100 and incubated with NOTCH1 for 1 h at room temperature. After washing with PBS, cells were incubated with goat-555 secondary anti-body for 1 h and stained with DAPI for 15 min. After washing with PBS, cells were examined under a fluorescent microscope .p ≤ 0.05.All experimental data were presented as the means ± SD. The significant differences between the groups were analyzed by Student’s t-test or one-way analysis of variance (ANOVA) followed by Newman-Keul’s test. Differences were considered to be significant at First, we investigated the changes of microRNA-139-5p in a model of osteoporosis. As shown in Then, we explored the mechanism of microRNA-139-5p on osteoporosis by up-regulating microRNA-139-5p expression using microRNA-139-5p mimics. There was an increase of microRNA-139-5p expression in BMSCs by over-expression of microRNA-139-5p, compared with the negative group . Over-exWe investigated the mechanism of microRNA-139-5p on osteogenic differentiation in BMSCs. As shown in To confirm whether NOTCH1 participated in the effects of microRNA-139-5p on osteogenic differentiation of BMSCs, si-NOTCH1 was used to inhibit NOTCH1 expression. Si-NOTCH1 suppressed NOTCH1 protein expression, and induced Wnt and β-catenin protein expression in BMSCs by down-regulation of microRNA-139-5p, compared with down-regulation of the microRNA-139-5p group . The inhWe also studied whether the suppression of Wnt/β-catenin participated in the effects of microRNA-139-5p on osteogenic differentiation in BMSCs. Wnt plasmid induced Wnt and β-catenin protein expression in BMSCs by down-regulation of microRNA-139-5p, compared with down-regulation of the microRNA-139-5p group . Activatet al. suggested that LINC00152 may function as an oncogene in oral squamous cell carcinoma and could be a potential therapeutic target in patients with  this disease [BMSCs, which derive from bone marrow, have self-renewing and multi-directional differentiation potential, and can differentiate into osteoblasts, chondrocytes, adipocytes and myocytes, respectively, under various induction conditions , 14. At  disease .et al. suggested that 25-hydroxycholesterol promotes RANKL-induced osteoclastogenesis through miR-139-5p [The osteogenic regulatory mechanisms of stem cells can be divided into intracellular and intercellular space regulatory mechanisms . Of themR-139-5p .et al. reported that Notch1 by miR-139-5p inhibits glioma metastasis and epithelial-mesenchymal transition [The recent research finds that NOTCH1 can also induce the directional osteogenic differentiation of MSCs, with the effect being remarkably stronger than the other members in the BMP family. The present study showed that the over-expression of microRNA-139-5p suppressed NOTCH1 protein expression, and induced Wnt and β-catenin protein expression in BMSCs. Li ansition .et al. showed that miR-139-5p elevates skeletal myogenic differentiation of human adult dental pulp stem cells (ADSCs) through the Wnt/β-catenin signaling pathway [RUNX2 is the recognized osteogenesis-associated transcription factor in the downstream BMPs, which plays a vital role in the development of the skeletal system . No oste pathway .BMP2 can regulate the expression of ALP through the Wnt signaling pathway, and it promotes the expression of ALP and osteogenic calcium deposition through the Wnt autocrine ring; in addition, the stable expression of β-catenin is required in the Wnt pathway in order to promote the expression of ALP . β-catenIn conclusion, our results indicate that microRNA-139-5p promotes osteogenic differentiation of BMSCs via Wnt/β-catenin signaling pathway by targeting NOTCH1, and moreover, that microRNA-139-5p may have potential clinical applications in osteogenic differentiation of BMSCs or osteoporosis.


Exposure to a heroin-associated conditioned stimulus can reactivate drug reward memory, trigger drug cravings, and induce relapse in heroin addicts. The amygdala, a brain region related to emotions and motivation, is involved in processing rewarding stimulus. Recent evidence demonstrated that disrupting the reconsolidation of the heroin drug memories attenuated heroin seeking which was associated with the basolateral amygdala (BLA). Meanwhile, neural functions associated with learning and memory, like synaptic plasticity, are regulated by glycogen synthase kinase 3 beta (GSK-3β). In addition, GSK-3β regulated memory processes, like retrieval and reconsolidation of cocaine-induced memory. Here, we used a heroin intravenous self-administration (SA) paradigm to illustrate the potential role of GSK-3β in the reconsolidation of drug memory. Therefore, we used SB216763 as a selective inhibitor of GSK-3β. We found that injecting the selective inhibitor SB216763 into the BLA, but not the central amygdala (CeA), immediately after heroin-induced memory retrieval disrupted reconsolidation of heroin drug memory and significantly attenuated heroin-seeking behavior in subsequent drug-primed reinstatement, suggesting that GSK-3β is critical for reconsolidation of heroin drug memories and inhibiting the activity of GSK-3β in BLA disrupted heroin drug memory and reduced relapse. However, no retrieval or 6 h after retrieval, administration of SB216763 into the BLA did not alter heroin-seeking behavior in subsequent heroin-primed reinstatement, suggesting that GSK-3β activity is retrieval-dependent and time-specific. More importantly, a long-term effect of SB216763 treatment was observed in a detectable decrease in heroin-seeking behavior, which lasted at least 28 days. All in all, this present study demonstrates that the activity of GSK-3β in BLA is required for reconsolidation of heroin drug memory, and inhibiting GSK-3β activity of BLA disrupts reconsolidation and attenuates heroin relapse. Opioid use disorder is a chronic recurrent brain disease caused by abnormal learning and memory patterns. And the symptom is the loss of substance use control. Drug-associated cues are important factors to promote the relapse to drug use. In addiction animal models, cues related to drug abuse can promote relapse . Once drSame as other types of memories , drug reA large amount of evidence shows that pharmacological interventions disrupting reconsolidation of drug-reward memory are effective in relapse prevention. Using a conditioned place preference (CPP) or self-administration (SA) animal model, propranolol or rapamycin has been shown to effectively interfere with reconsolidation of drug memory and attenuate drug-seeking behavior .Glycogen synthase kinase-3 (GSK-3), a serine/threonine-protein kinase expressed widely in the mammalian brain, has essential roles in physiological activities like development, cell cycle, or apoptosis . GSK-3β However, whether the inhibition of GSK-3β in BLA could disrupt reconsolidation and prevent drug seeking and relapse in heroin SA animal model remains unknown. In our present study, we inhibited the activity of GSK-3β in the BLA to identify the effects of GSK-3β on reconsolidation of heroin cue memory in SA animal model. Amygdala plays a critical role in both cue-associated learning and the expression of cue-induced relapse of drug-seeking behavior , and alsWe housed the Sprague Dawley rats  five-per-cage in a 26°C and 60% humidity room and the cycle was 12-h- light/dark (8 a.m.–8 p.m.). Food and water were provided ad libitum. For the animal better adapted to operator, we performed a 5-day grasp-stroking adaptation procedure 3 min per day on experimental animals before the surgeries. All animal procedures and operations were carried out with the approval of the Xiangya Hospital Ethics Committee, Xiangya Hospital . At the dark phase (8 a.m.–8 p.m.), the experiments were performed.Surgery was performed when the rats’ weight reaches 300–320 g. We used the sodium pentobarbital  to anesthetize rats. The right jugular was exposed by surgery, then inserted an aseptic catheter into it . The catGuide cannulae was implanted 1 mm above the BLA or CeA bilaterally after the rats (300–320 g) were anesthetized by using the sodium pentobarbital . And the coordinate of BLA  were theThe injection of SB216763 was based on a previous report by some minor changes . The BLAFollowing the study of n = 11) were excluded from the experiments: five rats died in intravenous surgery and six rats can’t form a stable heroin self-administration.We trained rats to adapt the intravenous heroin self-administration (0.05 mg/kg/infusion) for 10 days. In the training sessions, rats have been trained for three 1-h daily training sessions and every training session was separated by 5 min. The training of rats using a fixed ratio 1 (FR1) reinforcement program up to 40 s. In 10-day training program, rats were received three 1-h heroin infusions intravenously (0.05 mg/kg/infusion) per day, each infusion separated 5 min. All heroin infusions were completed by an injection pump loading a 10 ml syringe. Each training companied a house light illuminated until the end. When rats were at the left nosepoke (active), the maneuver results in an intravenous heroin infusion with a 5 s tone-light cue synchronization. However, the inactive nosepokes had no consequences. To prevent animals’ from death due to overdose, the drug infusions degree was restricted to 20 times/h . Some raAfter the drug self-administration session, a 3-h daily nosepoke extinction training was followed. In extinction training (Experiments 1–4), the nosepoke behavior of rats to the sensor resulted in no programmed consequences like heroin intravenous delivery, tone-light cues. When at the last three self-administration sessions, the number of active nosepoke responding decreased at least 80% compared with the self-administration, the nosepoke extinction session was ended.In Experiments 1, 2, and 4, the heroin-associated memories were reacted by performing a 15 min retrieval trial. The conditions of the retrieval session were the same as the SA training session, except that when rats got the “active” sensor, no reward heroin was infusion.In Experiments 1, 3, and 4, a cue extinction session, which 3-h per day, was performed on rats. The conditions of the cue extinction session were the same as that SA training session. But after the cue (tone/light) rendering, there was no heroin intravenous delivery.After the SB216763 or vehicle administration , rats got rest for 24 h and then returned to the SA training context and the reinstatement test was performed. In this session, the number of active and inactive nosepokes was recorded for 1 hr. The test condition was the same as the heroin memory retrieval trial.A low dose of heroin  was delivered to rats 5 min earlier before the session started, then put rats in the SA training context. Through the two sensors, the number of two type nosepokes (“active” and “inactive”) was recorded during the test. The reinstatement test lasted for 1 h. The test condition was the same as the heroin memory retrieval trial.In this test, after 28 days of the withdrawal phase, two types of nosepokes (“active” and “inactive”) were recorded for 1 h. The test condition was the same as the heroin memory retrieval trial.Through 10 days of heroin SA training, nosepoke extinction training was followed in the same apparatus for 10 consecutive days. After nosepoke extinction training, rats were allowed a rest for 24 h, then rats were received a 15-min conditioned stimulus (CS) retrieval session. After CS exposure, the rats were divided into four groups: (1) Intracranial injection of vehicle into BLA (0.5 μL/side) immediate after the retrieval trial (BLA+vehicle); (2) Intracranial injection of SB216763 into BLA (0.5 μL/side) immediate after a 15 min retrieval test (BLA+SB216763); (3) Intracranial injection of vehicle into CeA (0.5 μL/side) immediate after a 15 min retrieval test (CeA+vehicle); (4) Intracranial injection of SB216763 into CeA (0.5 μL/side) immediate after a 15 min retrieval test (CeA+SB216763). On Day 23, the rats were performed a cue-induced reinstatement test to explore the effect of SB216763 on heroin drug memory. Subsequently, the two-day cue extinction session was carried out. On Day 26, heroin priming-induced reinstatement was tested in rats .After CS exposure, the rats were divided into four groups: (1) Intracranial administration of vehicle into BLA (0.5 μL/side) immediate after a 15 min retrieval trial (BLA+vehicle); (2) Intracranial injection of SB216763 into BLA (0.5 μL/side) immediate after a 15 min retrieval trial (BLA+SB216763); (3) Intracranial injection of the vehicle into CeA (0.5 μL/side) immediate after a 15 min retrieval trial (CeA+vehicle); (4) Intracranial injection of SB216763 into CeA(0.5 μL/side) immediate after a 15 min retrieval (CeA+SB216763). Rats were received a cue-induced reinstatement test after a 24 h rest. After 28 days of abstinence, to access the long-term effect of SB216763 on heroin-seeking behavior, a spontaneous recovery test was carried out .The experimental procedure was the same as Experiment 1, except that no CS exposure session to reactivate heroin cue memories .The rats were received treatment of SB216763 delayed for 6 h after the retrieval session, and other parts of the experiment were the same as Experiment 1 .post-hoc test in each experiment (see Results). All values of experiments were presented as mean ± SEM and data analysis was done on GraphPad, v.9.0. p values < 0.05 were considered statistically significant.We used the repeated-measures ANOVAs to analyze the data with the between-subjects factor of treatment condition and within-subjects factor of test condition, followed by Tukey’s N = 10) or SB216763 (N = 11) and groups of CeA treated with vehicle (N = 10) or SB216763 (N = 11) which were shown by the heroin infusion numbers . In cue-induced reinstatement test, the post-hoc test revealed a reduction significantly in active side nosepokes of BLA+SB216763 group compared to that BLA+vehicle (p < 0.001)  = 1.187, p = 0.2895; administration condition: F = 0.2169, p = 0.6467; trial condition × administration condition interaction: F = 0.0156, p = 0.9020;  = 101.4, p < 0.0001; administration condition: F = 0.0052, p = 0.9432; trial condition × administration condition interaction: F = 0.0140, p = 0.9063;  = 0.7022, p = 0.4125; administration condition: F = 0.1727, p = 0.6824; trial condition × administration condition interaction: F = 0.0065, p = 0.9368;  = 51.16, p < 0.0001; administration condition: F = 9.702, p = 0.0057; trial condition × administration condition interaction: F = 12.55, p = 0.0022]; the post-hoc test showed a significant reduction of BLA+SB216763 group in drug-seeking compared with BLA+vehicle group in heroin priming-induced reinstatement test (p < 0.0001)  = 0.0450, p = 0.8342; administration condition: F = 0.0123, p = 0.9130; trial condition × administration condition interaction: F = 0.0736, p = 0.7891;  = 67.29, p < 0.0001; administration condition: F = 0.0369, p = 0.8498; trial condition × administration condition interaction: F = 0.2259, p = 0.6400;  = 0.0218, p = 0.8841; administration condition: F = 0.1738, p = 0.6814; trial condition × administration condition interaction: F = 0.0218, p = 0.8841; In active nosepokes, BLA+vehicle and BLA+SB216763 groups were significantly different in cue-induced reinstatement test . In the cue-induced reinstatement test, the post-hoc test shown a significant reduction in active side nosepokes of BLA+SB216763 group compared to that BLA+vehicle (p < 0.001)  = 1.212, p = 0.2854; administration condition: F = 0.0327, p = 0.8585; trial condition × administration condition interaction: F = 0.2860, p = 0.5993;  = 53.73, p < 0.0001; administration condition: F = 0.1503, p = 0.7028; trial condition × administration condition interaction: F = 0.0223, p = 0.8830;  = 1.683, p = 0.2110; administration condition: F = 0.0485, p = 0.8281; trial condition × administration condition interaction: F = 0.3407, p = 0.5666;  = 29.44, p < 0.0001; administration condition: F = 13.79, p = 0.0016; trial condition × administration condition interaction: F = 15.39, p = 0.0010]. During the spontaneous recovery test, a post-hoc test showed a significant reduction of BLA+SB216763 group in drug-seeking compared with BLA+vehicle group (p < 0.0001)  = 1.688, p = 0.2103; administration condition: F = 2.840, p = 0.1092; trial condition × administration condition interaction: F = 0.0604, p = 0.8086;  = 74.83, p < 0.0001; administration condition: F = 0.1074, p = 0.7469; trial condition × administration condition interaction: F = 0.027,p = 0.8714;  = 0.0110, p = 0.9176; administration condition: F = 0.1554, p = 0.6981; trial condition × administration condition interaction: F = 0.6468, p = 0.4318; Consistent with the results of Experiment 1, in the cue-induced reinstatement test, we found that nosepokes of groups differed from each other significantly in active side of BLA+vehicle and BLA+SB216763 groups [main effect of trial condition: F< 0.001)  left, bu 0.0001)  left butThus, the results suggest that immediate post-CS BLA SB216763 treatment rather than CeA can reduce subsequent cue-induced heroin seeking and last at least 28 days.N = 10) or SB216763 (N = 10) and of CeA administrated vehicle (N = 10) or SB216763 (N = 10) had no differences in acquisition session [main effect of acquisition time: F = 37.72, p < 0.0001; administration condition: F = 0.0764, p = 0.9724; acquisition time × administration condition interaction: F = 0.1869, p > 0.9999;  = 127.0, p < 0.0001; administration condition: F = 0.1659, p = 0.9187; extinction time × administration condition interaction: F = 0.4249, p = 0.9953; We tested whether SB216763 affecting subsequent heroin seeking depends on CS retrieval by using four rats groups. When heroin acquisition and extinction training were finished, rats were received BLA or CeA SB216763 treatment with no CS exposure . During  = 67.15, p < 0.0001; administration condition: F = 0.0008, p = 0.9778; trial condition × administration condition interaction: F = 0.0415, p = 0.8409;  = 0.0816, p = 0.7784; administration condition: F = 0.0184, p = 0.8937; trial condition × administration condition interaction: F = 0.0816, p = 0.7784;  = 78.74, p < 0.0001; administration condition: F = 0.0068, p = 0.9353; trial condition × administration condition interaction: F = 0.1212, p = 0.7317;  = 1.561, p = 0.2275; administration condition: F = 0.0312, p = 0.8618; trial condition × administration condition interaction: F = 0.0285, p = 0.8678;  = 61.05, p < 0.0001; administration condition: F = 0.0232, p = 0.8806; trial condition × administration condition interaction: F = 0.1465, p = 0.7064;  = 0.3709, p = 0.5501; administration condition: F = 0.0075, p = 0.9318; trial condition × administration condition interaction: F = 0.2887, p = 0.5976;  = 58.06, p < 0.0001; administration condition: F = 0.0397, p = 0.8444; trial condition × administration condition interaction: F = 0.0209, p = 0.8866;  = 0.1848, p = 0.6724; administration condition: F = 2.064, p = 0.1679; trial condition × administration condition interaction: F = 0.1118, p = 0.7420; Cue-induced reinstatement test was shown that no significant differences were found in nosepokes of active side [main effect of trial condition: FThus, the results indicate that the CS retrieval of drug-associated memory determines the effect of SB216763 on subsequent heroin seeking and when SB216763 is used alone, there will be no influence on the heroin seeking of the subsequent cue-induced and heroin-primed reinstatement test.N = 10) or SB216763 (N = 10) and of CeA administrated vehicle (N = 10) or SB216763 (N = 10) had no differences in heroin self-administration acquisition [main effect of acquisition time: F = 32.37, p < 0.0001; administration condition: F = 0.0423, p = 0.9882; acquisition time × administration condition interaction: F = 0.1182, p > 0.9999;  = 155.5, p < 0.0001; administration condition: F = 0.0613, p = 0.9798; extinction time × administration condition interaction: F = 1.120, p = 0.3139; Finally, we employed whether SB216763 treated out of the reconsolidation time window would suppress the following heroin seeking . During  = 73.20, p < 0.0001; administration condition: F = 0.0001, p = 0.9922; trial condition × administration condition interaction: F = 0.0675, p = 0.7979;  = 0.3240, p = 0.5763; administration condition: F = 0.0135, p = 0.9088; trial condition × administration condition interaction: F = 0.2073, p = 0.6543;  = 67.32, p < 0.0001; administration condition: F = 0.1421, p = 0.7106; trial condition × administration condition interaction: F = 0.0610, p = 0.8079;  = 0.0001, p = 0.9944; administration condition: F = 0.3902, p = 0.5405; trial condition × administration condition interaction: F = 0.0182, p = 0.8943;  = 77.38, p < 0.0001; administration condition: F = 0.1286, p = 0.7241; trial condition × administration condition interaction: F = 0.0481, p = 0.8289;  = 0.0473, p = 0.8304; administration condition: F = 0.2022, p = 0.6583; trial condition × administration condition interaction: F = 0.0241, p = 0.8783;  = 94.30, p < 0.0001; administration condition: F = 0.1377, p = 0.7149; trial condition × administration condition interaction: F = 0.0955, p = 0.7611;  = 0.3672, p = 0.5525; administration condition: F = 0.0108, p = 0.9184; trial condition × treatment condition interaction: F = 0.2189, p = 0.6458; In cue-induced reinstatement test, the nosepokes did not significantly differ in the active side [main effect of trial condition: FThe results suggest that the effect of SB216763 on the heroin seeking of the following cue-induced and heroin-primed reinstatement test is time-specific.via an intravenous heroin SA procedure. The main findings are following: (1) Immediate post-CS SB216763 treatment in BLA rather than CeA can reduce subsequent cue-induced and heroin-primed reinstatement of heroin seeking. (2) The effect of SB216763  immediately after retrieval session, which reactivated the heroin cue memory, on cocaine-seeking behavior continued for at least twenty-eight days; (3) SB216763 administration into BLA delayed 6 h or without the retrieval have no effect on the cue-induced and heroin priming-induced reinstatement test, suggesting that the effects of SB216763 on drug-seeking behavior are retrieval-dependent and time-specific. The results demonstrated that the activity of GSK-3β in BLA is required for reconsolidation and intra-BLA injection of SB216763 disrupts memory reconsolidation, suggested that inhibiting GSK-3β activity of BLA disrupts reconsolidation and attenuates heroin relapse.In our present study, we examined the effects of SB216763, a GSK-3β inhibitor, on heroin-seeking behavior and relapse via disrupting reconsolidation  or unconditioned stimulus (the drugs). Disrupting the reconsolidation after drug memory retrieval effectively blocks the association between drug-related cues and drugs, thus producing a forgetting effect and reducing drug-seeking behavior. Memory retrieval activates GSK-3β in the BLA, and reconsolidation was impaired by systemical administration of a GSK-3 inhibitor before memory retrieval in rats . We havelidation .Our present study complements the role of GSK-3β in heroin self-administration model. We show that inhibiting GSK-3β activity by injection of SB216763 into the BLA, but not the CeA, immediately after the retrieval reduced the drug-seeking behavior. In addition, some studies have shown that the GSK-3β in the hippocampus regulates learning and memory ability. Targeted downregulation of GSK-3β in the ventral hippocampus disrupting the formation of cocaine CPP and the spatial memory . OverexpThe amygdala, associated with emotion and motivation, plays roles in processing rewarding environmental stimulus . Recent GSK-3β is widely expressed in the brain and involved in fundamental brain functions like neurogenesis, neurotransmitter signaling , circadivia a rats heroin SA paradigm. After heroin cue memory retrieval, injection of SB216763 into BLA impairs reconsolidation of the heroin cue memory, effectively reduces the heroin seeking behavior in rats, and these effects lasted at least 28 days. Moreover, the reconsolidation window may be an important determinant to reduce heroin seeking and relapse. Our present study identifies that GSK-3β inhibitors may have the potential therapeutic value in the treatment of heroin addiction.In summary, the present study demonstrated the effects of GSK-3β on the reconsolidation of drug reward memory The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Xiangya Hospital Ethics Committee, Xiangya Hospital .HL and YX: conceptualization. YZ and YX: data curation. TH and ZZ: writing – original draft preparation. YX and QL: writing – review and editing. HL: supervision and funding acquisition. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


USH1C gene encodes the scaffold protein harmonin which organizes protein networks due to binary interactions with other proteins, such as all USH proteins. Interestingly, only the retina and inner ear show a disease-related phenotype, although USH1C/harmonin is almost ubiquitously expressed in the human body and upregulated in colorectal cancer. We show that harmonin binds to β-catenin, the key effector of the canonical Wnt (cWnt) signaling pathway. We also demonstrate the interaction of the scaffold protein USH1C/harmonin with the stabilized acetylated β-catenin, especially in nuclei. In HEK293T cells, overexpression of USH1C/harmonin significantly reduced cWnt signaling, but a USH1C-R31* mutated form did not. Concordantly, we observed an increase in cWnt signaling in dermal fibroblasts derived from an USH1CR31*/R80Pfs*69 patient compared with healthy donor cells. RNAseq analysis reveals that both the expression of genes related to the cWnt signaling pathway and cWnt target genes were significantly altered in USH1C patient-derived fibroblasts compared to healthy donor cells. Finally, we show that the altered cWnt signaling was reverted in USH1C patient fibroblast cells by the application of Ataluren, a small molecule suitable to induce translational read-through of nonsense mutations, hereby restoring some USH1C expression. Our results demonstrate a cWnt signaling phenotype in USH establishing USH1C/harmonin as a suppressor of the cWnt/β-catenin pathway.Human Usher syndrome (USH) is the most common form of hereditary combined deaf-blindness. USH is a complex genetic disorder, and the pathomechanisms underlying the disease are far from being understood, especially in the eye and retina. The  These USH1 genes encode for proteins of diverse protein families such as motor proteins, transmembrane proteins, and scaffold proteins (The human Usher syndrome (USH) is the most common form of inherited combined deaf-blindness with a prevalence of 1:6,000 to 1:10,000 in human . USH is s/USH1C) . USH1 isproteins , which aproteins .USH1C gene   at the end of the C-terminal domain to the PDZ1 and PDZ3 domains of harmonin (USH1C is truncated due to the R31* or the R80Pfs*69 (The scaffold protein harmonin is encoded by the  276904)  which cob, and c . Althougpithelia . Harmonipithelia  that arepithelia . Recentlharmonin  as previharmonin . When US80Pfs*69  this parβ-catenin is a multitasking and evolutionary conserved protein with dual major functions: it is not only an essential component of cadherin-based cell-cell adhesion complexes in adherent junctions (AJ) but also the key effector of canonical Wnt (cWnt) signaling in the nucleus , two kinases, GSK3b (glycogen synthase kinase 3b) and CK1 (casein kinase 1), and the protein phosphatase PP2A. Cytoplasmic β-catenin molecules are phosphorylated by the activity of the destruction complex and subsequently targeted for ubiquitination and proteosomal degradation  family. TCF/LEF proteins (hereafter referred to as TCF) bind to DNA but have a limited ability to activate transcription by their own. Binding of β-catenin leads to the formation of two-membered TCF/β-catenin transcriptional activators that convert Wnt signals into transcription of specific cWnt target genes (Binding of Wnt ligands to Fz (Frizzled) receptors and the co-receptors LRP5/6 (low density lipoprotein receptor-related protein) at the plasma membrane triggers molecular events leading to inhibition of the destruction complex. Activated receptor complexes recruit Dvl (dishevelled) through direct binding which induces formation of the LRP-associated Wnt signalosomes . Dvl, inactivity . Unphospet genes .β-catenin is much more complex, as briefly described above  of β-catenin can inhibit its degradation promoting the translocation of β-catenin into the nucleus and transcription of cWnt target genes  patient-derived cells. Conclusively, we provide first molecular and functional evidence for a role of cWnt signaling in the development in human USH.Here, we provide several lines of evidence that the USH1C protein harmonin is a suppressor of the cWnt signaling pathway by interacting with β-catenin_K49 (lys49)  commonly used for the analyses of β-catenin specifically acetylated at the residue 49 , rabbit polyclonal anti-harmonin (H3)  was used as a control. The pMS`BC-β-catenin plasmid was used for the expression of human β-catenin  was used expressing the human harmonin a1 isoform or a mutant harmonin R31* cDNA. The nonsense mutation R31* was introduced into the wild-type construct by -catenin .Ataluren  was dissolved in dimethyl sulfoxide . For treatment, 5 μg/mL and 10 μg/mL of Ataluren were used. DMSO (0.2%) served as control.2. Transfection of cells was performed using GeneJuice®  according to manufacturer’s instructions. Ataluren was applied 6 h after transfection. Cells were used for luciferase assays 48 h after Ataluren treatment.HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with Glutamax containing 10% heat-inactivated fetal bovine serum (FBS)  at 37°C in 5% COUSH1C mutations c.91C>T; p.(R31*) and c.238dupC; p.(R80Pfs*69) (USH1CR31*/R80Pfs*69) . We analysed dermal fibroblasts of a male patient genetically diagnosed for USH1 with biallelic 0Pfs*69) , as wellβ-catenin [β-cat-stimulation (β-catS)] (β-catenin (pMS`BC_β-catenin human) or alternatively, by culturing cells for 24 h in Wnt conditioned media [Wnt medium stimulation (WMS)] ]  transfecn (WMS)] . Wnt conβ-catenin signaling pathway activity was performed using the Dual-Glo® Luciferase Assay System  according to the manufacturer’s protocol. Briefly, HEK293T cells were seeded into a 96-well plate and after 24 h co-transfected with TopFlash (Firefly luciferase) for determining the activity of TCF/LEF dependent Firefly luciferase expression, and pRL-TK (Renilla luciferase) for normalization of the transfection efficacy, pcDNA-Dest47 vectors, namely pcDNA-Dest47-Harm_a1_GFP, pcDNA-Dest47-Harm_R31*_GFP, pcDNA-Dest47_GFP and additionally β-catenin (pMS`BC_β-catenin human) for stimulation. Alternatively, cells were stimulated with Wnt conditioned media 24 h post-transfections  for 15 min . Fixed cvia Fiji.For lysis, cells were incubated with Triton X-100 lysis buffer  containing protease inhibitor cocktail  and sonified. Cell lysates samples were mixed with SDS-PAGE loading buffer  and separated on self-made 12% or 15% polyacrylamide gels, first for 5 min at 110 V, then at least for 60 min at 180 V using the Mini-PROTEAN System . For Western blot (WB) analyses, proteins were transferred to polyvinylidene difluoride membranes  in blotting buffer  SDS, 20% (v/v) methanol) for 75 min at 100 V using the Mini Trans-Blot Electrophoretic Transfer Cell . After blocking the membrane with Applichem blocking reagent  for 1–2 h at room temperature, immunoreactivities were detected by applying primary and appropriate secondary antibodies, Alexa Flour 680 (Invitrogen) or IR Dye 800 , employing the Odyssey infrared imaging system  and processed ® agarose beads  were used for immunoprecipitation assays according to the manufacture’s protocol. In brief, GFP-tagged USH1C/harmonin proteins as well as β-catenin were expressed in HEK293T cells for 48 h. For lysis, cells were incubated with Triton X-100 lysis buffer  containing protease inhibitor cocktail  and sonified. Cells were lysed as described above and 10% of total cell lysate was separated for input fraction. Beads were equilibrated with 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA dilution buffer and incubated with remaining lysates of β-catenin and GFP-tagged harmonin or GFP only for 2 h at 4°C under constant rotation. After washing of beads with dilution buffer, precipitated proteins were eluted with Laemmli buffer and analyzed on SDS-PAGE (12% polyacrylamide gels) followed by Western blot (WB).Magnetic GFP-Trapin-situ proximity ligation assay Duolink PLA probes anti-rabbitPLUS, anti-mouseMINUS, and Detection Reagent Red  as previously described in . Objectives used were ×40 and ×63. Western blots were analyzed using the Odyssey infra-red imaging system . Image processing was done with ImageJ/Fiji software .β-catenin_K49 intensity in immunofluorescence staining in fibroblasts was performed using a script in ImageJ/Fiji software (β-catenin_K49 intensity in this area (µm2). PLA signals were identified with an ImageJ Fiji script by measuring the number of particles in the region of interest (ROI) processed picture. Pearson correlation coefficient was defined via Fiji using the colocalization Coloc 2 plugin (https://imagej.net/plugins/coloc-2). For Western blot analysis, relative band intensities were normalized to the relative band intensities of the corresponding control. Quantification of results was performed in MS-Excel using Student’s t-test . Error bars are represented as standard deviation. At least three independent experiments were performed. The significance levels were set when p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).Analysis of software . Brieflyopen(“...”);run;selectWindow(“Image001DMEM_ctr_ch03.tif”); run(“8-bit”);setAutoThreshold(“Default dark no-reset”);setOption;run(“Convert to Mask”); run(“Watershed”);run;close; selectWindow(“Image001DMEM_ctr_ch01.tif”);roiManager(“Show None”);roiManager(“Show All”);roiManager(“Measure”);run(“Flatten”);open(“...”);run(“8-bit”);run;//run(“Brightness/Contrast...”);run(“Apply LUT”);setAutoThreshold(“Otsu dark no-reset”);//run(“Threshold...”);//setThreshold;setOption;run(“Convert to Mask”);run;run(“Flatten”);® . After 24 h medium was changed to fresh DMEM or Wnt medium. The next day, cells were harvested and centrifuged in PBS. Cell pellets were resuspended in 1.25 packed cell volume of hypotonic MC buffer  and left on ice for 15 min. For lysis, a 1-mL syringe with G25 needle was used. For separation of nucleus and cytosol, lysed cells were centrifuged at 1,500 g for 5 min. The supernatant was taken (cytoplasmic fraction) and the nucleus pellet was incubated in Triton X-100 lysis buffer  containing protease inhibitor cocktail  and sonified. Protein lysates were separated by SDS-PAGE gel electrophoresis (15% polyacrylamide gels), followed by Western blotting as described above. Before blocking, blots were incubated and stained with Revert 700 total protein Stain pack  for visualizing of total protein amount.The isolation of cytosol and nucleus fractions was performed as described for the first steps by . BrieflyUSH1CR31*/R80Pfs*69 (USH1C) patient-derived and healthy control fibroblast cell lines were used. Human fibroblasts were maintained in either DMEM 10% FBS  or Wnt-3A conditioned medium  for 24 h at 37°C, 5% CO2 before RNA isolation. RNA isolation was performed using the RNeasy Mini Kit by following the manufacturer’s instructions . Subsequent RNA library preparation and whole transcriptome RNAseq were conducted by Novogene using the Illumina NovaSeq 6000 Sequencing System . Following quality control, genes with an adjusted p-value <0.05 were considered as significantly differentially expressed . Differentially expressed Wnt signaling pathway and Wnt target genes were identified by manual literature research -terms Wnt pathway (GO:0016055) and canonical Wnt pathway (GO:0060070) (http://www.heatmapper.ca/08.12.2022)  . For tho12.2022) . AdditioIn silico prediction tools were used to assess whether amino acid substitutions after translational read-through of the R31*-mutant USH1C/harmonin protein are expected to have an impact on the protein. All variants were denoted based on the NCBI reference sequence for USH1C . Variant classification was assessed using the following computational in silico tools: PolyPhen-2 and tools integrated into Alamut Visual Plus v.1.1 (Sophia Genetics) using default settings. PolyPhen-2 (Polymorphism Phenotyping v2) was used, which predicts the possible impact of an amino acid substitution on the structure and function of a human protein using straightforward physical and comparative considerations (https://agvgd.iarc.fr/index.php) (http://sift.bii.a-star.edu.sg/)   . SIFT (Sedu.sg/) . Mutatioer.org/) .β-catenin has been identified in a yeast two-hybrid screen by  in its very C-terminal tail to the PDZ1 and PDZ3 domains of harmonin  in HEK293T cells. Fluorescence microscopy revealed positive harmonin-β-catenin PLA signals in the cytoplasm and the nucleus indicating close proximity of both proteins  signaling pathway and its transcriptional activity is highly regulated by the formation of the β-catenin destruction complex (β-catenin (β-catenin_K49) and inhibits the formation of the β-catenin destruction complex and its degradation. Moreover, the acetylation promotes the nuclear translocation of β-catenin_K49 and thereby the activation of the cWnt signaling pathway ] (β-catenin [β-cat-stimulation (β-catS)]  was almost absent due its degradation by the activity of the β-catenin destruction complex . Such β-catenin filament bundles have been described previously in nuclei of MDKC cells overexpressing β-catenin ]  or by ovβ-catS)] . We subsractions . Confoca complex . However nucleus . Anti-GF-catenin . Analysiβ-catS) in both the cytosol and the nucleus fractions. However, when we calculated the ratio of harmonin in cellular fractions and total protein, we observed significantly more harmonin in the nuclear fraction after Wnt stimulation, via WMS as well as β-catS  in HEK293T cells in which the cWnt signaling was stimulated by overexpression of β-catenin (β-catS). The quantification of Western blots revealed that the ratio of β-catenin_K49/β-catenin was significantly reduced in harmonin overexpressing cells compared to control transfected cells , its harmonin a1_R31* (Harm-R31*) version or GFP alone, respectively and the relative firefly luciferase activity was determined (β-catenin (β-catS) led to drastic increases of the relative firefly luciferase activity, ∼10-fold and over 40-fold, respectively, indicating the high sensitivity of the assay and strong cWnt pathway activations under both stimulation conditions. Harm-a1 overexpression completely extinguished the relative firefly luciferase activity under conditions of stimulation by Wnt medium and significantly reduced the increase from 40-fold to less than 3-fold when cWnt signaling was stimulated by β-catenin. In contrast, the truncated Harm-R31* version reduced the relative firefly luciferase activity nearly to half under conditions of stimulation by Wnt medium compared to the GFP control and no significant difference in the relative luciferase activity between Harm-R31* and GFP controls was observed when cWnt signaling was stimulated by β-catenin. Taken together, our results obtained with the cWnt luciferase assay confirm that USH1C/harmonin suppresses cWnt signaling in HEK293T cells.To verify these promising results, we next analyzed cWnt activity using a cWnt signaling luciferase assay sensing of TCF/LEF transcription factor activity . For this HEK293T cells were transfected with Dual-Glotermined . WithoutUSH1CR31*/80Pfs*69 (hereafter referred to as USH1C fibroblasts) and a healthy control individual  in control medium and Wnt conditioned medium (WMS), respectively, and examined their transcriptomes by RNAseq.Next, we aimed to validate our results obtained in HEK293T cells in USH1C patient-derived dermal fibroblasts cells, which have recently been established as a cellular model of USH disease for translational research . In humaUSH1CR31*/80Pfs*69 mutations on transcriptome level, we additionally examined DEGs of healthy and USH1C fibroblasts. Consistent with the previous findings, WMS led to an increased amount of DEGs. Specifically, we were able to identify 3,265 DEGs , including 31 Wnt target genes when comparing healthy and USH1C Wnt stimulated fibroblasts . Moreovβ-catenin. We demonstrate that USH1C/harmonin deficiency in USH1C patient-derived fibroblasts significantly alters not only the expression of Wnt target genes but also of genes related to cWnt signaling pathway. Finally, we proof that the transitional read-through drug Ataluren restores the cWnt phenotype in HEK293T cells and patient-derived fibroblasts bearing an early nonsense mutation in USH1C. The novel role of USH1C/harmonin in the control of cWnt signaling identified here opens new avenues of research to unravel the pathomechanism of USH, identify new therapeutic targets, and evaluate treatment options in patient-derived cell models.In conclusion, we provide several lines of evidence that the USH1C protein harmonin is a potent suppressor of cWnt pathway by interacting with the cWnt pathway coactivator


This study is aimed to unravel the status of local and circulating β-catenin in different primary bone tumors and its relevance to tumor types, severity, and chemotherapy. The β-catenin mRNA expression level and the expression of the protein (intensity level) were evaluated in tumor tissue and peripheral blood mononuclear cells of 150 patients with different types of primary bone tumors  using Real-Time PCR and immunohistochemistry. The β-catenin mRNA expression level and the expression of the protein were increased in bone tumors which was positively correlated with the tumor malignancy. Amongst osteosarcoma, Ewing's Sarcoma, chondrosarcoma, osteochondroma, Giant Cell Tumor, and exostosis tumors, the osteosarcoma, and Giant Cell Tumor groups showed the highest level of β-catenin expression. The β-catenin expression in malignant bone tumors was significantly correlated with tumor grade, size, metastasis, tumor recurrent, and the level of response to chemotherapy. A similar pattern of β-catenin gene expression and its association with tumor characteristics was detected in the patient's peripheral blood cells. The simultaneous increase in the expression of the β-catenin gene and protein in tumor tissue and in circulating blood cells and its relationship with tumor severity indicates the possible promoting role of β-catenin in primary bone tumor pathogenesis. The bone tumor incidence rate comprises at least two distinct peaks of an individual's life in the second decade and late adulthood2. The dynamic of bone tissue-dependent on the tissue’s physiological and pathological condition which makes it susceptible to cellular re-arrangement, tumor cell formation, and tumor cell implantation3. Despite recent improvements regarding primary bone tumor diagnosis and therapeutic strategies, still nonspecific symptoms besides late patient referring and early detection failing are prominent drawbacks that cause cancer-induced morbidity and mortality4. The Wnt/β-catenin pathway received considerable attention as effective cellular signaling which is associated with the pathogenesis of several cancers 5. Various cellular functions likewise cell proliferation, polarity, fate, migration, renewal, regeneration as well as organogenesis during embryonic development and tissue homeostasis are under the regulation of the canonical Wnt/β-catenin pathway6. Beta-catenin is the transcriptional activator that belongs to the cadherin protein complex and controls the expression of several targets involved in cell development and fate. The β-catenin accumulation or mutation was detected in some malignancies likewise hepatocellular carcinoma, endometrial carcinoma, gastric adenocarcinoma7 Interestingly, the relevance of canonical Wnt signaling machinery was reported to be involved in bone homeostasis8. It was reported that activation of β-catenin in osteocytes enhances the osteoclasts and osteoblasts' activity resulted in bone gain and is required for osteocyte viability and maintaining normal bone mass10. Additionally, deregulation of β-catenin can be involved in bone–related diseases likewise bone metabolic disease and bone tumors11. It was revealed that the Wnt signaling pathway is implicated in Ewing's Sarcoma biology and might be regulated by the EWSR1-FLI1, the major oncogene of Ewing's Sarcoma and involved in tumor migration or differentiation13. Based on the mechanistic studies regarding osteosarcoma pathogenesis, it seems that the canonical Wnt signaling pathway is involved in osteosarcoma progression possibly through naked cuticle homolog 2 (NKD2) which negatively regulates the Wnt/β-catenin pathway14.Primary bone tumors are considered as non-frequent mesenchyme-originated solid tumors which affected individuals at all age rangesDespite promising evidence, questions might be raised due to the status of β-catenin as a pivotal mediator of the Wnt canonical pathway in human samples with diverse bone tumors. How is the expression pattern of β-catenin in prevalent malignant bone tumors? Does the β-catenin expression differ in Ewing's Sarcoma compared to osteosarcoma or chondrosarcoma? How is the β-catenin expression pattern in benign bone tumors? Do chemotherapy treatments affect β –catenin expression in the site of the tumor and peripheral blood? Does the patient's peripheral blood as an invasive source of the patient’s sample reflect the local β-catenin expression in tumors? Is it possible that the patient's peripheral blood serves as a predictor of β-catenin expression pattern in bone cancer? Our study is designed to peruse the abovementioned remarks.Our data indicated that the β-catenin expression increases significantly in bone tumors compared to normal bone tissues P < 0.0001) which is shown in Fig.  which isThe expression level of β-catenin was assessed in PBMCs of the patients with different types of bone tumors due to the feasibility of liquid biopsy efficiency for early diagnosis. Based on our data, a significant increase in the mRNA expression level of β-catenin was detected in patients with malignant tumors (0.5695 ± 0.02443) compared to patients with benign tumors (0.1408 ± 0.01348) indicating a considerable fourfold increase (P < 0.0001)  leads to the proteosomal degradation of β-catenin and the non-phosphorylated β-catenin accounts as the active protein that mediates target gene transcription18. To determine whether the increased β-catenin mRNA expression level in bone tumor tissues is accompanied with its elevated protein level, the β-catenin protein level was assessed using immunohistochemistry in several types of malignant and benign bone tumors. The un-phosphorylated form of β-catenin that is known as the active form of this protein19 was assessed in the current study. The protein level of β-catenin was evaluated and scored by the pathologist and data are summarized in Table β-Catenin, as an important mediator of the canonical Wnt signaling pathway, plays a decisive role in cell adhesion, movement and proliferation. Cytoplasmic accumulation and nuclear translocation of β-catenin indicates that the Wnt canonical pathway is activatedTo better determine how the local and circulating mRNA expression level of β-catenin, the protein expression, and patient's demographic features are related, the correlation of β-catenin expression with the aforementioned factors was evaluated for each tumor type separately and the results are presented in Supplementary Tables 20. Also, the lower expression of β-catenin in Ewing’s Sarcoma tumors was detected compared to osteosarcoma tumors indicating that the overall impact of Wnt mediators in Ewing’s Sarcoma is driving differentiation rather than proliferation 13. It was shown that up-regulation of β-catenin is a genetic feature that is associated with metastasis of tumor cells since the decrease in β-catenin expression and transcription level caused attenuation of the metastasis-associated phenotypes of breast cancer cells 21. Also, the association of β-catenin/Wnt pathway knock-down with increased apoptosis restoration is postulated in tumor cells22. Besides, it was proven that β-catenin is involved in the regulation of cancer stem cell marker genes that play a functional role in cell renewal ability and stemness features that all together suggest that β-catenin may critically act in fostering chemo resistance of tumor cells 24. It was shown that utilizing β-catenin transcription inhibitors can sensitize osteosarcoma cell lines to the chemotherapeutic agents revealing the impactful role of β-catenin in tumor response to the chemotherapy treatment 25. Notably, activation and/or reactivation of tumor cells after the remission period accounts for a challenging and perilous issue in cancer management which might be occurred in up to 65% of bone cancers 26. In accordance, to get insights into the correlation of β-catenin expression level with tumor metastasis, recurrent and chemo resistance, our data revealed that an increase in the expression level of β-catenin was associated with tumor metastasis, recurrent and poor response to chemotherapy in osteosarcoma, and Ewing's Sarcoma which is in line with previous evidence. In the current study, patients with chondrosarcoma tumors had no history of chemotherapy and none of them had recurrent tumors; while an increase in β-catenin expression level was also correlated with tumor metastasis in the chondrosarcoma group. Also, based on our data, the elevated expression of β-catenin protein (intensity level) along with other variables increases the risk of metastasis and tumor grade of bone tumors. Also, it was found that β-catenin suppression/ degradation inhibited cell proliferation and invasion as well as induced apoptosis in renal cell carcinoma 27 and lung cancer cell 28. Moreover, multiple lines of evidence indicate that activation of the β-catenin pathway promotes cell proliferation, invasion, and migration through different mechanisms such as Long noncoding RNAs regulation 29 and interaction with histone demethylase 30. In the current study, an increase in the β-catenin expression was associated with malignant tumor grade and size indicating the possible positive role of β-catenin in enhancing primary bone tumor proliferation and growth. In support of our data, it was delineated that the interaction of β-catenin with forkhead box protein M1 (FOXM1) mediates osteosarcoma cell growth and metastasis 31. To clarify whether the β-catenin expression pattern can be affected by tumor type; the most prevalent benign bone tumors such as Giant Cell Tumor, osteochondroma, and hereditary multiple exostosis were also included in this survey and our data revealed a significant increase in β-catenin expression level in benign bone tumors compared to normal bone tissues, also malignant tumors expressed a higher level of β-catenin significantly compared to benign bone tumors;. Based on our data, Giant Cell Tumors expressed a higher level of β-catenin compared to the other benign bone tumors and showed a significant association between tumor size and β-catenin expression level. The relevance of β-catenin function in bone hemostasis, metabolism, and turnover has been illustrated in previous studies. It was shown that Wnt canonical pathway is involved in the osteogenesis process specifically via sclerostin and cell surfaces proteins such as ZNRF3 and RNF4332. Also, activation of β-catenin/Wnt pathway caused elevation of osterix and osteoprotegerin expression level which promoted osteoblast differentiation and osteoclast suppression, respectively 33. Several findings support the fact that peripheral blood can be served as a possible circulating source of biomarkers and a less invasive diagnostic route that can reflect the immune system behavior and response in pathologic situations 34. Also, as a possible circulating source of cancer cells/cancer stem cells that have been released in the blood, perusing peripheral blood isolated cells beside tumor tissues might provide a better and more comprehensive picture of molecular targets and immune cells alterations in a pathological situation such as cancer 35. In accordance, the mRNA expression assessment of β-catenin in PBMCs of patients with malignant and benign bone tumors revealed an increase in the expression level of β-catenin in malignant compared to benign tumors. The same expression pattern of β-catenin was observed in tumor and PBMCs of each malignant tumor type compared to normal bone tissues, also patients with osteosarcoma and Giant Cell Tumors expressed a higher level of β-catenin compared to the rest of tumor types in their group, respectively. Based on our data, the circulating level of β-catenin in malignant tumors was correlated with tumor metastasis, recurrent, and grade. Also, based on the fact that the mRNA level might be influenced by different genetic and epigenetic processes that can cause loss of protein expression 36, in the current study, the local active form of β-catenin protein was also assessed in malignant and benign tumors. The overexpression of β-catenin protein was detected in malignant compared to benign bone tumors and the β-catenin expression was associated with tumor grade, metastasis, recurrent, and response to therapy. Therefore, the β-catenin gene and protein expression revealed consistency in different types of primary bone tumors also the simultaneous increase in the expression of β-catenin in tumor site and patient's PBMCs indicates the possible role of β-catenin as a diagnostic marker in bone cancer. The correlation of β-catenin expression with patients' age was only detected in PBMCs of patients with chondrosarcoma which can be explained by the fact that in the chondrosarcoma group, all of the participants were at the same age range (> 20 years). Taken together, assessing the simultaneous local and circulating expression pattern of β-catenin in diverse types of malignant and benign bone tumors at both gene level and protein expression underlines the possible relevance of β-catenin to primary bone cancer pathogenesis, however, is required further mechanistic studies and clinical studies with more patients. Also, survival analysis on patients with varying degrees of tumor severity and response to treatment in relation to β-catenin expression can also provide valuable information.It is taken for granted that bone is avascular and fibrous tissue with the ability to change its cell profile and is a suitable substrate for the placement of circulating tumor cells. On the other hand, common and traditional therapeutic approaches for primary bone tumor treatments limit the efficacy and efficiency of the definitive treatment of this disease. Accordingly, genomic characterization of tumors besides unraveling intracellular pathways that are involved in tumor pathogenesis, might pave the way to introduce actionable and impressive molecular targets as new therapeutic goals. β-catenin as the main mediator of the canonical Wnt pathway, gained considerable attention due to its involvement in important aspects of cellular processes such as cell invasion, migration, proliferation, and differentiation. Also, the regulatory role of β-catenin in the epithelial-mesenchymal transition (EMT) process, as a critical step of tumor cell invasion, emphasizes its important role in cancer cell fate. Considering the fundamental and decisive role of β-catenin in tumor cell formation and tumor progression and the fact that β-catenin is a functional readout of the Wnt pathway that can be modulated and targeted, its local and circulating expression level was investigated in six types of primary bone tumors with different patient’s clinic pathological features, in the current study. Based on our data, a considerable increase in the mRNA and protein expression of β-catenin was observed in primary bone tumors compared to bone normal tissues. The increase in the β-catenin expression was apparent in osteosarcoma, Ewing's Sarcoma, and chondrosarcoma compared to the matched tumor margins, in which the higher expression of β-catenin was detected in osteosarcoma tumors compared to the other malignant bone tumors. In line with our evidence, it was shown that β-catenin expressed more in osteosarcoma compared to osteochondroma 37. A pair of the tumor and marginal tissue samples were taken from all the patients who were subjected to surgery at Shafa Orthopedic Hospital and were kept immediately at − 80 for later assays. The tumor and normal bone tissue collection and processing were followed as in our previous study38. For evaluation of circulating factors, the 6 ml peripheral blood was taken from the patients as well as healthy controls and used for peripheral blood mononuclear cell (PBMC) separation. The patients and healthy subjects were matched as a matter of age and sex. In the current study three types of common malignant bone tumors including osteosarcoma as a deadly form of bone tumors, Ewing's Sarcoma as a small round cell tumor and chondrosarcoma as a malignant tumor consisting of chondrocytes were included39. Also, three types of benign bone tumors including Giant Cell Tumors which are also known as osteoclastomas, osteochondroma as benign single chondrogenic lesions and hereditary multiple exostosis as a genetic disorders consisting of multiple osteochondromas were included40. The clinic-pathological features of patients are summarized in Table 41. In the current survey, 63.2% of patients with osteosarcoma and 66.7% of patients with Ewing's Sarcoma showed poor response to the chemotherapy treatments. Also, 33.4% of patients with osteosarcoma and 26.9% of Ewing's Sarcoma patients had recurrent tumors while none of the patients with chondrosarcoma had recurrent tumors. The difference between chondrosarcoma and the other two malignant tumor groups as a matter of age (P < 0.0001) tumor grade (P = 0.035), and chemotherapy received/not received (P < 0.0001) was statistically significant. Also, the difference between osteosarcoma and chondrosarcoma as a matter of tumor size (0.029) was statistically significant, while the rest of the features showed no specific difference between malignant bone tumor groups. Additionally, Table The number of 150 bone tumor tissues, 150 tumor margins, and 150 peripheral drops of blood taken from the same patients with bone tumor as well as 32 healthy blood samples were enrolled in the current study with local ethical approval and informed consent. The project was ethically approved by the ethics committee of the Vice president of research of the Iran University of Medical Sciences with the ethics committee code: IR.IUMS.REC 1395.95-04-30-29364. Written informed/Informed consent was obtained from all participants before enrolling in the study and for those patients whose age was less than 18 years, consent was submitted from their legal guardian and after approval and signing of consent, the patient was included in the survey. Signed informed consent of participants will be provided upon request. Also, all procedures and methods were performed following the relevant guidelines and regulations with the ethical standards of the responsible committee on human experimentation in Iran University of Medical Sciences and in accordance with the Helsinki DeclarationTo separate peripheral blood mononuclear cells (PBMCs) from the collected blood of patients and healthy controls, Ficoll-Hypaque  was utilized with subsequent density gradient centrifugation. The separated cells were washed with phosphate-buffered saline (PBS) and counted by hemocytometer. An equal number of PBMCs was applied for further assessment.−ΔCt) method was applied for the analysis of gene expression.Evaluation of β-catenin gene expression level was assessed using quantitative real-time PCR. Accordingly, the tumor, adjacent noncancerous tissues, and PBMCs were subjected to the RNA extraction via Trizol  based on the manufacturer’s instructions. Briefly, 700 µl of Trizol lysis reagent was used to lysis and homogenize bone tissues, after a sufficient incubation period, chloroform was added to each tissue homogenate for subsequent phase separation. The aqueous phase was collected and mixed with isopropanol. Following incubation and centrifugation, the remaining pellet containing RNA was washed with 75% ethanol, air-dried, and dissolved in RNase-free water. The Nanodrop spectrophotometer (Nanodrop Technologies) was applied for evaluating the quantity and quality of the RNA extracted from each sample. The cDNA synthesis was implemented using the PrimeScript First Strand cDNA Synthesis Kit  fro1 µg of the extracted RNA based on the manufacturer’s instructions. The SYBR Premix Ex Taq II  was used for assessment of β-catenin gene expression which was performed in Applied Biosystems Step One Plus, Real-time system . For detection of β-catenin gene level, the specific primers were designed and the beta-actin expression level was considered as a housekeeping gene. The sequence and characters of primers were as: Catenin forward primer: 5′-TCACTCCTCCTAATGGCTTG-3′, Catenin reverse primer: 5′-GTTGCTGCCAGTGACTAACA-3′ (Tm = 58), beta-actin forward primer: 5′-GAT CTC CTT CTG CAT CCT GT-3′, beta-actin reverse primer: 5′-TGG GCA TCC ACG AAA CTA C-3′ (Tm = 57). The primer’s specificity was determined by melting curve analysis for each amplified product. The running PCR program was as 1 cycle at 95 °C for 5 min following 40 cycles at 95 °C for 5 s, 55 °C for 20 s and 60 °C for 35 s. The PCR products were measured and approved their product length by electrophoresis. The comparative CT  embedding medium was applied for preparing the frozen tissue blocks following incubation at − 27 °C. Tissue sections were prepared using cryotome and tissue slides were exposed to the washing buffer, H2O2 solution, Triton5%, and blocking buffer before staining by β-catenin primary antibody. The non-phosphor- β-catenin rabbit monoclonal antibody  was used in a dilution of 1:700, and tissue slides were incubated at a required time. The anti-rabbit IgG HRP-conjugated secondary antibody  were used to visualize the primary antibody binding. For final dehydration and subsequent mounting, ethanol and xylolin were applied. The stained tissues were observed and scored by pathologists who were blinded to the data. The staining intensity method was applied to determine the immunoreactivity of β-catenin antibody and it was indicated as the absence of immunoreactivity; weak intensity; moderate intensity; and strong intensity in five different hot spot areas.The Hematoxylin and eosin (H&E) histological staining was applied to identify bone cancerous and non-cancerous tissues and determine tissue composition, affinity and interaction to the dye−ΔCt) method was used wherein ΔCt indicates the subtract of β-catenin Ct from the Ct of the endogenous gene (β-Actin). To determine whether the results of β-catenin gene expression are normally distributed in patients  also in the PBMC of the healthy group, the Kolmogorov–Smirnov analysis was applied before analyzing the difference in expression of β-catenin between different groups. Due to the normal distribution, the difference in the expression level of β-catenin was analyzed using t-student test between tumor and margin, malignant and benign tumors, malignant and benign tumor subgroups and their matched normal margins also malignant tumors with different tumor features  (Results are illustrated in Figs. For gene expression analysis, the comparative CT 2−ΔCt methSupplementary Tables.


Transplantation of neural progenitor cells (NPCs) derived from human‐induced pluripotent stem cells (hiPSCs) is one of the promising treatment strategies for motor neuron diseases (MNDs). However, the inefficiency in committed differentiation of NPCs in vivo limits its application. Here, we tried to establish a potential therapeutic strategy for MNDs by in vivo directional differentiation of hiPSCs engineered with motor neuron (MN) specific transcription factors and Tet‐On system.We engineered hiPSCs with three MN‐specific transcription factors and Tet‐On system. The engineered cells were directly transplanted into immunodeficient mice through subcutaneous, intra‐spinal cord and intracerebroventricular injections. Following doxycycline (Dox) induction, teratoma formation, and motor MN differentiation were evaluated.Ngn2, Isl1, and Lhx3 was controlled by a drug‐inducible transgenic system. These cells showed normal pluripotency and proliferative capacity, and were able to directionally differentiate into mature motor neurons (MNs) and NPCs with high efficiency in spinal cords and cerebral lateral ventricles under the induction of Dox. The grafts showed long‐term survival in the recipient mice without formation of teratoma.We generated genetically engineered hiPSCs, in which the expression of The induced mature MNs and NPCs were expected to replace the damaged endogenous MNs directly, and play a role of de novo stem cell stock for long‐term neuron damage repair, respectively. Therefore, in vivo directional differentiation of the hiPSCs engineered with MN‐specific transcription factors and Tet‐On system via Dox induction could be a potential therapeutic strategy for MNDs with high efficacy and safety. Ngn2, Isl1, and Lhx3 controlled by Tet‐On system and one anti‐apoptosis gene Bcl‐xL. The genetically modified hiPSCs survived and directionally differentiated into motor neurons after transplantation in vivo. The genetically modified hiPSCs partially converted into neural progenitors, serving as a spare cell resource.Generation of hiPSCs with multi‐gene modifications, including motor neuron‐specific transcription factors  Owing to the difficulty of neuronal regeneration, clinical studies have failed to develop effective strategies to replace lost motor neurons (MNs). Stem cells have great potential for the replenishment of lost neuronal population in MNDs. Intensive efforts have been made in developing stem cell‐based strategies for MNDs. In particular, mesenchymal stem cells are being tested in early‐stage clinical trials due to their good safety and tolerability. However, mesenchymal stem cells cannot differentiate into neurons and have only shown limited beneficial effects in clinical studies.The in situ direct conversion of endogenous astrocytes to functional neurons by the ectopic expression of defined factorsNgn2, Isl1, and Lhx3, can efficiently induce functional MNs with mature electrophysiological properties from hESCs or hiPSCs.Ngn2, Isl1, and Lhx3 genes. The cells showed normal proliferative capacity and directional differentiation into MNs in doxycycline (Dox) medium in vitro. Further in vivo experiments revealed that these engineered hiPSCs could convert into MNs and NPCs whether in subcutaneous tissues, spinal cords, and cerebral lateral ventricles. These induced mature MNs may be used to directly replace the damaged endogenous MNs, and NPCs may provide de novo stem cell stock for long‐term neuron damage repair.Three MNs that induce transcription factors, namely, 22.1Ngn2‐Isl1‐Lhx3‐BSD (PB‐NIL) plasmid was described in De Santis et al.Bcl‐xL‐Luciferase‐GFP (PB‐BLG) plasmid was generated by inserting the sequences of Bcl‐xL (gene ID 397536) and firefly luciferase‐GFP  in the PB vector. pGL4.21 was purchased from Promega.The PB‐2.2NIL: transposase = 4:1 or PB‐NIL: PB‐BLG: transposase = 2:2:1) using Lipofectamine LTX (Invitrogen). After blasticidin  selection, the BSD‐positive colonies were collected with a pipette and used to generate two stable hiPSC lines (termed NIL‐hiPSCs and NILB‐hiPSCs). MN differentiation of NIL‐hiPSCs and NILB‐hiPSCs was induced by the addition of 1 μg/ml Dox (Sigma) in MN induction medium.HiPSCs were co‐transfected with transposable vectors and PiggyBac transposase  In vitro differentiation was evaluated by immunofluorescence staining using MN, NPC, and MNP markers; (ii) the functional characteristics of hiPSC‐derived MNs were determined by patch‐clamp recordings; and (iii) the pro‐survival capacity of 2.3NILB‐hiPSCs were directly transplanted into immunodeficient mice without in vitro differentiation through subcutaneous, intra‐spinal cord, or intracerebroventricular injections. Following Dox induction , teratoma formation and MN differentiation were evaluated as follows: (i) the survival of transplanted cells was tracked by bioluminescent imaging; (ii) tumorigenicity was determined by the haematoxylin and eosin (H&E) staining of three germ layers and expression of pluripotent markers; (iii) in vivo differentiation was evaluated by immunofluorescence staining using MN, NPC, and MNP markers; and (iv) the functional characteristics of hiPSC‐derived MNs were determined by brain and spinal cord slice electrophysiology.All animal care and experimental procedures were approved by the Ethical Committee on Animal Experiments at Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. 2.4p value of <0.05 was considered statistically significant.All statistics, including statistical tests, sample sizes, and types of replicates, were described in the figure legends. A Detailed experimental procedures are described in the Supporting Information Material 33.1NIL contains Dox‐inducible polycistronic genes Ngn2, Isl1, and Lhx3 (NIL) and constitutively expresses BSD resistance gene  and NILB‐hiPSCs (containing PB‐NIL and PB‐BLG). Both cell lines formed round colonies with clear margins similar to those of normal hiPSCs, and NILB‐hiPSCs were positive for EGFP  was markedly lower than that of NIL‐hiPSCs  and persistent outward currents (K+) in response to depolarizing voltage . The second experiment showed that the MNs from NIL‐hiPSCs had great susceptibility to glutamate, with only 46.43% ± 9.45% of the induced MNs surviving after glutamate treatment; meanwhile, up to 73.26% ± 7.60% of the neurons from NILB‐hiPSCs survived . The ectopic expression of Bcl‐xL conferred the pro‐survival ability of MNs, suggesting that induced MNs can survive and perform their function after transplantation in vivo. Thus, NILB‐hiPSCs were used for the following in vivo experiments.Two different in vitro models were used to determine the pro‐survival capacity of 3.3NILB‐hiPSCs were subcutaneously injected into immunodeficient mice to initially evaluate the differentiation direction of these engineered hiPSCs in vivo. In vivo imaging showed that the luciferase activity increased rapidly with time in the control group (without treatment of Dox)  for 5 days, the luciferase activity decreased in a time‐dependent manner, was almost undetectable at 6 weeks, and completely disappeared at 8 weeks post‐transplantation (w.p.t.) , that is, they were transplanted human cells. Many of the human cells expressed TUJ1  were positive for the neuronal marker TUJ1 . Further immunostaining showed that the grafts expressed mature MN markers, including MAP2 , HB9 , and ChAT  can simultaneously program hiPSCs into neurons and NPCs due to the intrinsic fluctuations in mmRNA and protein levels.Ngn2 to directly convert NILB‐hiPSCs into MNs under Dox induction. However, a small number of NILB‐hiPSCs might express a low level of Ngn2 with oscillatory‐like pattern under Dox induction, thus resulting in NPC formation. Brain development studies showed that neurogenesis declines with ageing.NILB‐hiPSCs could survive long‐term in vivo and may play the role of stem cell backup to fix injured neurons. This characteristic should be a potential advantage in curing chronical neurodegenerative diseases, such as ALS.In this work, NILB‐hiPSCs also developed a teratoma after their subcutaneous transplantation without Dox treatment. However, after Dox treatment for only 5 days, the destination was narrowed to neural cell lineage, and no tumour formation was noted in the subcutaneous tissues. The induced MNs gradually dwindled and ultimately disappeared at 8 w.p.t., which differed from the situation in the CNS. We speculate that this phenomenon probably occurred because the neurons could not adapt to the non‐neural microenvironment when transplanted into the subcutaneous tissues. Our results confirmed that the induced MNs survived well in the CNS for more than 20 weeks. The microenvironmental conditions of the transplant site may contribute to cell survival and function. Most of the transplanted cells initially died after being transplanted into the pathological environment. Pro‐survival strategies have been reported, including genetic modifications, cell preconditioning, and use of biomaterials.HiPSCs have a tendency to form a teratoma, which is the main concern of clinical practices. The generated Despite these beneficial potentials, the safety risks of engineered pluripotent cells are still the most important challenge for their clinical application. Overdose of Dox treatment may lead to side effects on gastrointestinal tract,Ngn2, Isl1, and Lhx3 factors in our system are replaced by other cell type‐specific transcription factors, then diverse cell types could be induced in vivo to treat corresponding organ failure or tissue loss. From this point of view, the developed novel stem cell‐based strategy may be applicable to MNDs and other diseases caused by cell loss.In summary, we describe a rapid, effective, and long‐lasting approach that generates an enriched population of MNs in vivo and should be valuable for the treatment of MNDs. Further studies are required to determine the therapeutic effects of these hiPSCs on small and large animal models of MNDs. A previous study showed that hiPSCs could be directly converted into a variety of specific human cells under the action of lineage‐specific transcription factors.Min Chen, Xia Wang, and Chuan Li performed experiments, analysed data, and wrote the manuscript. Ting Lan and Yuhui Wei performed animal experiments and collected samples. Chengcheng Tang, Xiaoqing Zhou, Alessandro Rosa, Renping Zhou, Xi Zheng, and Song Ang constructed the plasmids and revised the manuscript. Kun Zhang, Qingjian Zou, and Liangxue Lai designed and supervised the project, and wrote the manuscript.The authors declare no conflict of interest.Appendix S1 Supporting Information.Click here for additional data file.