HNF4α status which represses EMT, found in well-to-moderately differentiated HCCs with low AFP levels and good prognosis. HNF4α induces the expression of OATP1B3, which can be recognized as higher enhancement nodules in the hepatobiliary-phase of Gd-EOB-DTPA-enhanced MRI. On the other hand, Hyde phenotype is defined by high FOXM1 status which promotes EMT, found in poorly differentiated HCCs with high AFP levels, positive stem cell markers and poor prognosis. FOXM1 induces the expression of GLUT1, which can be recognized with FGD-PET/CT. HCC with activated Wnt/β-catenin signaling pathway. It could be hypothesized that the former should be mainly resistant to immune checkpoint inhibitors, while the latter would be immune activated and exhausted. In the future, the activation of the Wnt/β-catenin signaling pathway should be detected non-invasively and applied to the treatment of advanced HCC.Wnt/β-catenin mutations in HCCs have a dual phenotype. Jekyll phenotype is defined by high Combination therapy with immune checkpoint inhibitors (ICIs) and vascular endothelial growth factor inhibitors has been approved as a first-line treatment for unresectable hepatocellular carcinoma (HCC), indicating a critical role of ICIs in the treatment of HCC. However, 20% of patients do not respond effectively to ICIs; mutations in the activation of the Wnt/β-catenin pathway are known to contribute to primary resistance to ICIs. From this point of view, non-invasive detection of Wnt/β-catenin activation should be informative for the management of advanced HCC. Wnt/β-catenin mutations in HCC have a dual aspect, which results in two distinct tumor phenotypes. HCC with minimal vascular invasion, metastasis, and good prognosis is named the “Jekyll phenotype”, while the poorly differentiated HCC subset with frequent vascular invasion and metastasis, cancer stem cell features, and high serum Alpha fetoprotein levels, is named the “Hyde phenotype”. To differentiate these two HCC phenotypes, a combination of the hepatobiliary phase of gadolinium-ethoxybenzyl-diethylenetriamine (Gd-EOB-DTPA)-enhanced magnetic resonance imaging and fluoro-2-deoxy-D-glucose-PET/CT may be useful. The former is applicable for the detection of the Jekyll phenotype, as nodules present higher enhancement on the hepatobiliary phase, while the latter is likely to be informative for the detection of the Hyde phenotype by showing an increased glucose uptake. Cancer is often explained by new applications in cell-to-cell evolutionary biology. Neoplasms grow in a complex ecosystem, and natural selection is a powerful defense mechanism in cancer. The development of cancer is attributed to clonal selection; genetic mutations that favor cell proliferation are called driver mutations, and malignant neoplasms emerge by acquiring a growth advantage through cell proliferation . It has TERT) activation have been reported . . 16]. In the absence of Wnt, Axin functions as a scaffolding protein of the β-catenin pathway, specifically and efficiently promoting the phosphorylation of β-catenin by glycogen synthase kinase 3β (GSK-3β). Intracytoplasmic β-catenin binds to the central part of Axin, and APC binds to the regulator of the G protein signaling (RGS) domain, forming the Axin/APC complex. In the Axin/APC complex, multiple β-catenins are sequentially phosphorylated by GSK-3β, and phosphorylated β-catenins are successively degraded by the ubiquitin-proteasome system. Phosphorylation of Axin and APC enhances the phosphorylation of β-catenin . Wnt binding to the Frizzled/LRP trimeric co-receptors enhances the phosphorylation of the cytoplasmic tail of LRP5/6. These phosphorylation signals suppress the phosphorylation of β-catenin in a GSK-3β-dependent manner. The hypo-phosphorylated β-catenin escapes proteasome-mediated degradation and accumulates in the cytoplasm. The recruitment of Axin away from the destruction complex leads to stabilization and accumulation of β-catenin. Caveolin-dependent endocytosis of the Axin complex by the plasma membrane LRP6 receptor upon stimulation by Wnt3a is considered essential for β-catenin stabilization. In the nucleus, β-catenin binds to T-cell factor/lymphoid enhancing factor 1 (TCF/LEF1) to promote the transcription of Wnt target genes . R-spondβ-catenin, whose phosphorylation and degradation are blocked by Wnt canonical stimulation, enters the nucleus and binds to the TCF/LEF1 family of transcription factors to regulate the expression of target genes. In the absence of Wnt/β-catenin signaling, TCF acts as a repressor of Wnt/β-catenin target genes by forming a complex with the Groucho/TLE transcriptional co-repressor. Wnt signaling induces conflicting events, such as cell proliferation and terminal differentiation . In othec-myc and cyclin D1; second, genes related to body axis formation and organogenesis; third, genes related to the regulation of Wnt signaling, such as Wnt3a, Axin2, Frizzled, LEF1, TCF1, and LGR5.The Wnt/β-catenin signaling pathway is crucial for both normal development and carcinogenesis . More thSnail1, Snail2, and Twist are well-known Wnt target genes. Interactions between β-catenin and TGF-β signaling pathways mediate EMT [Snail and Twist are also targets of TGF-β signaling, which is one of the critical pathways of HCC formation [MMPs and VEGF, which are known to be involved in neovascularization and interact with cancer stem cell markers such as Sal-like protein 4 (SALL4), epithelial cell adhesion molecule (EpCAM), and the adhesion molecule E-cadherin.The transcription factors Snail (Snail1), Slug (Snail2), and Twist are the key inducers of EMT that are associated with tumor metastasis, and iate EMT   smaller than 30 mm. The results showed that in well-differentiated HCC, intranuclear accumulation of β-catenin was negatively correlated with mortality outcome, while in poorly differentiated HCC, intranuclear accumulation of β-catenin was a significant poor prognostic factor . AuthorsReports of canonical Wnt/β-catenin signaling activation and the malignant phenotype or prognosis of HCC have been confounded by this duality. Yamashita et al. reported that this paradox may be explained by the exclusive expression and activation of hepatocyte nuclear factor (HNF)4α and forkhead box M1 (FOXM1)  Figure . β-catenHNF plays an important role in the development of the liver. HNF4 is a member of the steroid hormone receptor superfamily, and HNF4 messenger RNA (mRNA) has been shown to be present in the kidney, intestine, and abundantly in the liver, but is absent in other tissues . HNF4α wAmong the hepatic transcription genes identified in previous studies on the transcriptional regulatory network in hepatocytes, HNF4α was also identified as one of the most important hepatic transcription factors. HNF4α is probably the most upstream mediator, acting as a master gene that regulates the transcription cascade to promote differentiation, proliferation, and morphogenesis of the hepatic lineage. It has been reported that Wnt/HNF4a interacts with LEF1 to affect liver zonation .HNF4α has also been reported to regulate EMT in HCC cells by inhibiting Wnt/β-catenin transcription and enhancing the localization of β-catenin in the cell membrane . ThroughARNTL, also known as BMAL1) gene [In a recent study, two HNF4α promoters were identified (P1 and P2), each of which expresses HNF4α transcriptional variants that are differentially expressed in human HCC and colon cancer . PromoteL1) gene . In normL1) gene . MiR-122L1) gene .It has been well described that green hepatoma, characterized by tumor cholestasis and showing a high-intensity nodule on Gd-EOB-DTPA-enhanced-MRI, generally shows a good prognosis and carries CTNNB1 mutations. OATP1B3, a transporter of bile acids, was found to be highly expressed in green hepatoma, and HCCs with high expression of OATP1B3 were negative for cancer stem cell markers, including cytokeratin-19 (CK19) and EpCAM .Gd-EOB-DTPA-enhanced MRI is a commonly used modality for the diagnosis of HCC. The liver-specific contrast agent used in MRI imaging maintains high diagnostic accuracy because the contrast agent is taken up by the hepatocytes via OATP1B3 expressed on the vascular side, indicating the potential of EOB-MRI as an imaging biomarker for predicting CTNNB1 mutations. Some studies indicated that the high expression of OATP1B3 was strongly correlated with a higher enhancement in hepatobiliary phase of Gd-EOB-DTPA-enhanced MRI ,8. The sThe vital role of the Wnt/β-catenin signaling pathway in the proliferation and self-renewal of normal cells and cancer stem cells is commonly accepted. Among HCCs with Wnt/β-catenin activation, some show an aggressive phenotype that is positive for EMT transcription factors and cancer stem cell markers, including CK19, EpCAM, and SALL4 ,31. In aFOX proteins are transcription factors that regulate embryonic development and tissue homeostasis, and they are highly conserved across species. There are 44 FOX family members in humans, which are subclassified into 19 classes (A to S) based on sequence resemblance of the DNA-binding forkhead domain . FunctioFOXM1 codifies for a proliferation-specific transcription factor that plays a critical role in cell cycle progression and initiation of cancer development. Deletion of FOXM1 in HCC inhibited tumor growth, indicating that FOXM1 is required for tumor progression [c-myc and cyclin D1. Cyclin D1-positive HCC has been reported to show an aggressive tumor phenotype and a poor prognosis, and it is reasonable to assume that the Wnt/FOXM1 axis promotes an aggressive phenotype of this subset [The master control gene gression . It has gression . Besidess subset  (Figure ARF tumor suppressor gene has been reported to play an important role in the pathogenesis of HCC, showing genetic alteration, hypermethylation of the promoter, and loss of heterozygosity in the 9p21 region [Continuous daily injections of a cell-penetrating ARF peptides in HCCs in mice with FOXM1 activity has also been shown to inhibit tumor cell proliferation and angiogenesis and significantly increase apoptosis within the HCC region . P19 ARF1 region . Other s1 region ,38,39,401 region .Intracellular energy metabolism is highly conserved in many organisms. Inhibition of energy metabolism pathways are involved in gene expression through epigenetic mechanisms. Various proto-oncogenes induce the expression of glucose transporters and glycolytic enzymes that lead to the elevation of the glycolytic system, indicating that tumor cells are highly dependent on the glycolytic system . In addiSLC2A1-AS1/STAT3 interaction [To date, HNF4α-negative and FOXM1-positive HCC with activated Wnt/β-catenin signaling, namely the aggressive phenotype, should be a candidate for systemic chemotherapy, including ICIs, but has not yet been effectively diagnosed using current imaging techniques. The promoter of FOXM1 contains a STAT3 binding site, and FOXM1 has been reported to regulate the transcription of GLUT1 through the eraction  (Figure eraction . In addieraction . Based oThe first-line therapy for unresectable HCC with Child-Pugh class A liver function is a combination of ICIs and VEGF inhibitors ,55. HoweActivation of canonical Wnt/β-catenin signaling, whose ligands are Wnt3a, Wnt1, and Wnt7, leads to two different phenotypes of HCC. One is attributed to the induction of HNF4α, which causes β-catenin activation in certain Hoshida S3 HCCs . In thisIn contrast, activation of FOXM1 by the canonical Wnt/β-catenin signaling pathway may result in Hoshida S2 HCC, which shows high AFP and stem cell features ,31 Figu. The macIn the non-canonical Wnt signaling pathway, binding of the weak trans-forming ligands Wnt5a, Wnt6, and Wnt11 promotes activation of the Wnt/TGF-β axis. The non-canonical Wnt/TGF-β signaling pathway is associated with the S1 subclass of Hoshida’s classification . This suIn a study of resected specimens, Wnt/β-catenin-positive HCC was reported to be around 30–40%, and OATP1B3-positive HCC recognized by the hepatobiliary phase of Gd-EOB-DTPA-enhanced MRI was reported to be around 15–20%, suggesting the possibility that a certain number of Wnt/β-catenin-positive, HNF4α-negative, FOXM1-positive HCC subsets exist. However, the study using resected specimens has a selection bias, in that patients with vascular invasion and multi-organ metastasis cannot not be subjected to surgical resection. The next challenge is to validate our findings in a variety of advanced HCC samples.HNF4α is a tumor suppressor gene that represses EMT, which is predominantly found in well-to-moderately differentiated HCCs and defines a subset with low AFP levels and good prognosis. It is a target gene of the Wnt/β-catenin pathway and induces the expression of OATP1B3 by co-expression, which can be recognized as a non-invasive nodule with high enhancement in the hepatobiliary phase of Gd-EOB-DTPA-enhanced MRI.On the other hand, HCCs with FOXM1 expression, located downstream of the Wnt/β-catenin pathway, are an aggressive and poor prognostic subset presenting high AFP levels, positive stem cell markers, poorly differentiated with EMT, and reduced E-cadherin expression. This subset lacks HNF4α expression and is not recognized as a nodule with higher enhancement by the hepatobiliary phase of Gd-EOB-DTPA-enhanced MRI. However, since it expresses GLUT1, it can be noninvasively recognized with FGD-PET/CT.It has been reported that HCC with an activated Wnt/β-catenin signaling pathway is primarily resistant to first-line immune checkpoint inhibitors. In the future, the activation of the Wnt/β-catenin signaling pathway should be accurately detected and applied to the treatment of advanced HCC for which biopsy specimens cannot be obtained.


Zinc-finger of the cerebellum 2 (Zic2) is widely implicated in cancers, but the role of Zic2 in tumorigenesis is bilateral. A recent study indicated that Zic2 could render colon cancer cells more resistant to low glucose-induced apoptosis. However, the functional roles of Zic2 in colon cancer and the underlying molecular mechanism remain elusive. Herein, we demonstrated that Zic2 was highly expressed in colon cancer tissues and correlated with poor survival. Knockdown of Zic2 inhibited colon cancer cell growth, arrested the cell cycle transition from G0/G1 to S phase, and suppressed tumor sphere formation in vitro; in addition, silencing Zic2 retarded xenograft tumor formation in vivo. Consistently, ectopic expression of Zic2 had the opposite effects. Mechanistically, Zic2 executed its oncogenic role in colon cancer by enhancing Wnt/β-catenin signaling. Zic2 directly binds to the promoter of Axin2 and transcriptionally represses Axin2 expression and subsequently promotes the accumulation and nuclear translocation of β-catenin. Meanwhile, Zic2 could activate Wnt signaling by interacting with β-catenin. Intriguingly, in HCT116 cells with intrinsic Ser45 mutation of β-catenin, which blocks the degradation-related phosphorylation of β-catenin by CK1, modified Zic2 expression did not affect the protein level of β-catenin. Altogether, our findings uncover a novel multilevel mechanism for the oncogenic activity of Zic2 in colon cancer and suggest Zic2 as a potential therapeutic target for colon cancer patients. Approximately 90% of CRCs have abnormal Wnt signaling activation, which plays important role in cancer stem cell (CSC) biology, tumorigenesis, and metastasis in CRC5. Nuclear translocation of β-catenin is considered the hallmark of the canonical Wnt pathway6. The disruption of the β-catenin destruction complex, consisting of CK18, GSK-3β10, Axin11, and APC12, results in the activation of Wnt signaling. Among the β-catenin destruction complex, AXIN2, playing crucial role in Wnt/β-catenin signaling pathway, participates in regulating cell proliferation, apoptosis, migration, and other important functions in the development of various cancers13. It is generally accepted that CK1, by binding to Axin2, initiates β-catenin degradation by phosphorylation at Ser45; subsequently, GSK-3β catalyzes the phosphorylation of β-catenin at more N-terminal residues 14, while APC can intensify the affinity of phosphorylases for β-catenin to accelerate β-catenin degradation in the cytoplasm via the ubiquitin-proteasome6. Dephosphorylated β-catenin accumulated in the cytoplasm can translocate into the nucleus and form complexes with member(s) of the TCF/LEF family to initiate transcription of downstream target genes, such as cyclin D1, CD44, c-Myc, and Lgr518. Mutations in APC19, β-catenin14, and Axin117 in CRC block the destruction complex’s affinity for β-catenin, which triggers aberrant accumulation of β-catenin. Among colon cancer cell lines, HCT116 has a special three-base deletion in the β-catenin encoding gene, CTNNB1, causing an in-frame deletion at Ser4520.Colorectal cancer (CRC) is the second most prevalent cancer and the fourth leading cause of cancer-related lethality globally23. As a transcription factor, Zic2 plays important role in cerebral formation in both animals and humans25. Mutations in Zic2 cause spina bifida and axial and limb bone malformation23. Likewise, Zic2 functions in tumorigenesis26, cancer stem cell renewal21, proliferation31, invasion, and metastasis32. However, the functions and mechanisms of Zic2 in various cancers are extraordinarily complicated and remain unclear29. In CRC, only one Zic2-related study has been published, in which Zhao et al. reported that Zic2 can render colon cancer cells more resistant to low-glucose-induced apoptosis31. However, how Zic2 regulates CRC tumorigenesis and progression remains elusive.Zinc-finger protein of the cerebellum 2 (Zic2), homologous to the Drosophila protein odd-paired, belongs to the Zic family of proteins, which contain highly conserved C2H2 zinc-finger motifsA tissue microarray of 180 pairs of primary CRC tissues was purchased from Shaanxi Kexin Biotechnology Co., Ltd (cohort I). Four hundred and three colon cancer tissue samples and paired normal colon tissue samples were randomly selected from CRC patients who had not received radiotherapy or chemotherapy before excision between January 2011 and December 2016 (cohort II). All the patients underwent surgery at the First Affiliated Hospital of Xi’an Jiaotong University. Informed consent forms were signed by all patients. Our study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Xi’an Jiaotong University.Lentiviral vectors with Zic2 shRNA or Zic2 overexpression were purchased from GeneChem Co., Ltd. . The target short hairpin RNA (shRNA) sequences were presented in Supplementary Table 33. The Zic2-stained  sections were divided into two groups (High and Low) based on scores of the staining between colon cancer tissues and adjacent colon normal tissues. Two pathologists evaluated all the specimens in a blinded manner.For IHC, the staining procedure was performed using the standard avidin-biotin complex method. The extent of positively stained cells was scored as previously described33.Total RNA was isolated from cells using TRIzol reagent . Complementary DNA (ctDNA) was synthesized by the PrimeScript RT Reagent Kit . Real-time PCR was conducted on an IQ5 instrument  using SYBR Green fluorescence signal detection assays  with primers list in Supplementary Table 33.For promoter analyses, a fragment of the Axin2 5′-flanking sequence (from −912 to +207 bp) and other truncated fragments were cloned into the pGL3.0 Basic Vector  to generate a Axin2 full promoter reporter construct and the truncated ones  according to the method of the manufacturer’s instructions2 atmosphere.DLD-1, HCT116, and SW480 cells  were authenticated by STR analysis . Cell lines were maintained in DMEM and McCoy’s 5A medium  supplemented with 10% FBS  and 100 units/ml of penicillin and streptomycin at 37 °C in a humidified 5% CO33. The Zic2-stained  sections were divided into two groups (High and Low) based on scores of the staining between colon cancer tissues and adjacent colon normal tissues. Two pathologists evaluated all the specimens in a blinded manner.For IHC, the staining procedure was performed using the standard avidin–biotin complex method. The extent of positively stained cells was scored as previously described3 cells per well, and cultured for up to 2 weeks to allow for spheroid growth. The medium was refreshed every 3 days.Cells were counted and plated in SFM  in ultralow attachment six-well plates  at a total of 1.5 × 102 and 37 °C on 96-well plates (100 μL/well). Ten microliters of CCK-8 reagent  was added to each well after 24, 48, 72, and 96 h, respectively. OD450 values were determined by a microplate reader.Cells were incubated at 5% COCells were plated into 60-mm plates and routinely cultured for 14 days. The cells were subsequently fixed with methyl alcohol for 15 min and stained with 0.1% crystal violet.After staining, the visible colonies were counted.Cells were harvested and fixed in 75% cold ethanol and stored at 4 °C overnight. The next day cells were centrifuged at 1500 rpm for 5 min. After treatment with RNase A at 37 °C for 30 min, cell pellets were further washed with PBS and stained with PI (10 μg/100 μl) for 10 min in the dark. The cell cycle was assessed with flow cytometry (Beckman Coulter).6) in logarithmic phase were sub-cutaneously injected into the right flanks of 5-week-old female BALB/c-nude mice . After 1 week post-injection, the length (a) and width (b) of the tumor were monitored using callipers every 3 days. The tumor volume (V) was calculated as follows: V = ab2/2. Three weeks later, the mice were sacrificed and the xenograft tumors were measured.The use of all animals in this study was approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Xi’an Jiaotong University. Tumor cells . Cell lysates containing 30 μg of total protein were then subjected to SDS–PAGE  and then transferred to PVDF membranes . The membranes were incubated with primary antibodies overnight at 4 °C , β-Catenin, Flag, and c-Myc, 1:1000 dilution; Cyclin D1 and GAPDH, 1:5000). The membrane was then washed six times with TBST buffer for 5 min each and incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Chemiluminescent HRP substrate  was added to visualize the protein bands. The antibodies against GAPDH were purchased from Santa Cruz , the antibodies against Zic2, Cyclin D1, CD44, Axin2, and APC were purchased from Abcam . The antibodies against active-β-Catenin(ser45), β-Catenin, and GSK-3β were purchased from Cell Signaling Technology , and the antibodies against Flag was purchased from Proteintech . Nuclear extract was prepared with the protocol in the Nuclear Extraction Kit .34 by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio  is more than 20%; (3) Removing reads whose unknown base (‘N’ base) ratio is more than 5%, afterward clean reads were obtained and stored in FASTQ format. The clean reads were mapped to the reference genome using HISAT2 (v2.0.4)35. Bowtie2 (v2.2.5)36 was applied to align the clean reads to the reference coding gene set, then expression level of gene was calculated by RSEM (v1.2.12)37. The heatmap was drawn by pheatmap (v1.0.8)38 according to the gene expression in different samples. Essentially, differential expression analysis was performed using the DESeq2(v1.4.5)39 with Q value ≤0.05. To take insight to the change of phenotype, GO (http://www.geneontology.org/) analysis of annotated different expressed gene was performed by Phyper (https://en.wikipedia.org/wiki/Hypergeometric_distribution) based on Hypergeometric test. The significant levels of terms and pathways were corrected by Q value with a rigorous threshold  by Bonferroni40.The sequencing data was filtered with SOAPnuke (v1.5.2)g for 20 min. Thermo Scientific Pierce Co-IP kit (Thermo Fisher Scientific) was used for Co-IP experiments. At last, obtained proteins were resuspended in 5× SDS sample loading buffer, heated to 100 °C for 8 min, and tested by 10% SDS–polyacrylamide gel electrophoresis (PAGE).Cells were lysed in Triton X-100 lysis buffer  containing phosphatase inhibitors and protease inhibitor , then the proteins extract were centrifugated at 12,000 × The cells were washed three times with PBS for 10 min each, fixed with 4% paraformaldehyde for 20 min, and permeabilized with 0.2% Triton X-100 for 10 min. After blocking with 5% bovine serum albumin (BSA) for 30 min at room temperature, the cells were incubated at 4 °C overnight with primary antibodies against Zic2 (1:100 dilution). The dishes were washed three times with PBS for 10 min each and then incubated with Alexa Fluor 594-conjugated secondary antibodies  for 1 h at room temperature. The nuclei were stained with DAPI (10 mg/ml) for 10 min. The samples were examined via microscopy  to analyze the expression and nuclear location of Zic2.P < 0.05 was defined as statistically significant.All data are presented as the mean ± standard deviation (SD). The chi-square test or ANOVA was used to analyze the differences among groups. Correlations were analyzed using Pearson linear-regression analysis. OS and RFS rates were plotted using the Kaplan–Meier method and compared with log-rank test. Multivariate statistical analysis was performed using a Cox regression model. All statistical analyses were performed using SPSS 22.0 software . To explore the role of Zic2 in colon cancer, we first analyzed the expression of Zic2 between colon cancer and normal colon tissues by bioinformatic analysis. The results showed that Zic2 transcript levels were upregulated in colon cancer compared with normal colon tissues in the GSE44706 mRNA microarray dataset Fig.  and TCGAP < 0.0001, Fig. To confirm the role of Zic2 proteins in colon cancer, we performed immunohistochemical (IHC) staining of 180 colon cancer samples via a tissue microarray  by western blotting and qRT-PCR analysis , including 422 downregulated and 508 upregulated genes in ectopically expressed Zic2 clones .A recent study indicated that Zic2 can render colon cancer cells more resistant to low-glucose-induced apoptosis by activating Wnt signalingsis Fig. . Ectopic16. In response to ectopic expression of Zic2, the expression of cyclin D1, CD44, and Lgr5 in DLD-1, HCT116, and SW480 cells was remarkably elevated at both the protein and mRNA levels  were elevated in response to ectopic expression of Zic2 in the DLD-1 and SW480 cell lines. Intriguingly, the levels of total β-catenin were not changed in the HCT116-Zic2 cell line. In addition, barely any active β-catenin protein could be detected in HCT116 cells due to the Ser45 mutation in this cell linenes Fig. . Taken tGO molecular function analysis suggested that Zic2 might execute its biological function in colon cancer via DNA/protein binding and transcription regulator activity  reporter construct and three other truncated constructs were constructed and transfected into DLD-Zic2 and SW480-Zic2 cells and control cells Fig. . The lucTo further determine whether Axin2 is involved in Zic2-mediated Wnt signaling activation, we stabilized Axin2 using IWR-1-endo and further investigated its impact on the functions of Zic2 Fig. . TreatinTo validate the function of Zic2 in vivo, Zic2-overexpressing colon cancer cells and negative control cells were subcutaneously injected into nude mice. Three weeks later, we measured the weight of xenograft tumors and found that ectopic expression of Zic2 significantly augmented the weight of xenograft tumors Fig. . By IHC 30, clear cell renal cell carcinoma41, oral squamous cell carcinoma42, and epithelial ovarian cancer43. In our study, a similar conclusion was drawn in colon cancer, and this is the first analysis of the relationship between Zic2 and prognosis using human colon cancer samples. However, Zic2 is barely expressed in breast cancer, in which high Zic2 expression predicts better survival. The reason for its positive relationship with prognosis in breast cancer is because in this cancer, Zic2 directly represses STAT3 transcription29. However, in most cancers, Zic2 acts as a transcriptional activator promoting tumorigenesis31. Intriguingly, we demonstrated for the first time that Zic2 acts as both a transcriptional coactivator and transcriptional repressor in colon cancer. Zic2 directly represses Axin2 transcription, which results in the disruption of the β-catenin destruction complex and the subsequent accumulation of active β-catenin in SW480 and DLD-1 cells. More intriguingly, ectopic expression of Zic2 in HCT116 cells did not change the level of active β-catenin, which might be due to the in-frame deletion at Ser45 in HCT116 cells20. Given that Zic2 activated Wnt/β-catenin signaling in HCT116 cells, we speculated that there exists another mechanism of Zic2-mediated Wnt signaling activation. As expected, we confirmed that Zic2 interacts with β-catenin in SW480 and DLD-1 cell lines besides the HCT116 cell line31. Zic2 might enhance Wnt/β-catenin signaling in colon cancer via chromatin remodeling45. Collectively, in addition to directly repressing Axin2 transcription, Zic2 also interacts with β-catenin to activate Wnt signaling in colon cancer. Our study revealed multilevel regulation of Wnt signaling by Zic2 in colon cancer.Zic2 is upregulated and correlates with poor survival in various cancers, such as hepatocellular carcinoma31. However, limited downstream target genes affected by the Zic2-Wnt/β-catenin axis have been identified, with only Glut1 being reported so far31. In the present study, we identified that Zic2 enhances Wnt signaling and initiates the transcription of several novel downstream target genes, including cyclin D1, CD44, and Lgr5, which promote proliferation and stemness in colon cancer cells.A previous study reported that Zic2 enhances Wnt signaling and plays crucial roles in tumorigenesis, and the same was found in our study31 but not in breast cancer29. Zic2−/− mice display a normal rate of proliferation46. Interestingly, Ishiguro et al. reported that phosphorylation of Zic2 at Ser200 decreases the interaction between Zic2 and RNA helicase in mice, which can suppress the transcriptional activation potential of Zic247. However, posttranslational modification and mutation of Zic2 cannot be excluded as reasons for our current findings, and the influence of these factors remains to be further evaluated. The discrepancy in its effects in different cancers makes it difficult to define the role of Zic2 in proliferation. In our study, ectopic expression of Zic2 promoted colon cancer proliferation by activating cyclin D1 transcription, in addition to cell cycle transition from G0/G1 to S phase in vitro. Chandrasekaran et al. reported that silencing Zic2 expression in benign prostate epithelial cells inhibited anchorage-independent spheroid and tumor formation by repressing CD44, Sox2, and Notch1 expression, indicating that Zic2 might be closely associated with self-renewal properties26. A similar conclusion was made in liver cancer stem cells21. Herein, we identified for the first time that Zic2 accelerates sphere formation in vitro. Zic2 upregulated CD44 and Lgr5, well-known CSC markers52, via Wnt/β-catenin signaling. Our study also revealed that Zic2 has effects on the maintenance of colon CSCs.A role of Zic2 in proliferation has not been conclusively demonstrated. Zic2 promotes proliferation in various cancersIn summary, we demonstrated that Zic2 is upregulated in colon cancer samples and that high Zic2 expression correlates with unfavorable survival of colon cancer patients. Our results indicate that Zic2 promotes the proliferation and tumorigenesis of colon cancer. Zic2 functions in colon cancer, at least in part, by enhancing Wnt/β-catenin signaling via collaboration with β-catenin and repressing Axin2. The above results suggest that the inhibition of Zic2 might be a potential therapeutic strategy for colon cancer patients.Supplementary Table1-5.docxSupplemental Figure LegendSupplementary. Figure 1Supplementary. Figure 2Supplementary. Figure 3Supplementary. Figure 4Supplementary. Figure 5Supplementary. Figure 6


Background: Seeking novel and effective therapies for gastric precancerous lesions (GPL) is crucial to reducing the incidence of gastric cancer. Ginsenoside Rb1 (GRb1) is a major ginsenoside in ginseng and has been proved to possess multiple bioactivities. However, whether GRb1 could protect against GPL and the underlying mechanisms have not been explored.Methods: We evaluated the effects of GRb1 on gastric precancerous lesions in rats on macroscopic, microscopic and ultramicroscopic levels. Then, an antibody array was employed to screen differential expression proteins (DEPs). Validation for the targeting DEP and investigation for the possible mechanism was conducted using immunohistochemistry, qRT-PCR, TUNEL apoptosis assay, immunoprecipitation and immunoblotting.Results: GRb1 was found to reverse intestinal metaplasia and a portion of dysplasia in the MNNG-induced GPL rats. The antibody array assay revealed seven DEPs in GPL rats as compared to control rats . Among the DEPs, β-catenin, beta-NGF and FSTL1 were significantly down-regulated after GRb1 administration. Our validation results revealed that enhanced protein expression and nuclear translocation of β-catenin were present in animal GPL samples. In addition, analysis of human gastric specimens demonstrated that β-catenin up-regulation and nuclear translocation were significantly associated with advanced GPL pathology. GRb1 intervention not only decreased protein expression and nuclear translocation of β-catenin, but interfered with β-catenin/TCF4 interaction. Along with this, declined transcriptional and protein expression levels of downstream target genes including c-myc, cyclin D1 and Birc5 were observed in GRb1-treated GPL rats.Conclusion: GRb1 is capable of preventing the occurrence and progression of GPL, which might be contributed by diminishing protein expression and nuclear translocation of β-catenin and interfering with β-catenin/TCF4 interaction. Gastric cancer still remains a major cancer worldwide, ranking fifth for incidence and fourth for mortality globally in the latest published global cancer statistics . Gastricginseng) is the major constituent screened from Weipixiao  were obtained from Chengdu Dashuo Experimental Animal Co., Ltd., Chengdu, China (SCXK-2020-030). This experiment was conducted in TCM Regulating Metabolic Diseases Key Laboratory of Sichuan Province, Hospital of Chengdu University of Traditional Chinese Medicine. Animal welfare considerations taken and all procedures were approved by the Institutional Animal Care and Use Committee .Formalin-fixed, paraffin-embedded samples of GPL gastric mucosa (94 cases) and normal gastric mucosa (85 cases) were provided from the Hospital of Chengdu University of TCM , and retrospectively analyzed. The pathological diagnosis of each sample was performed by two independent senior pathological staffs in the Department of Pathology. This study was approved by the Institutional Review Board of the Teaching Hospital of Chengdu University of TCM   and written informed consent was obtained from all participants for being included in the study.N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was purchased from Tokyo Chemical Industry CO., LTD., Japan (cat. no. M0527); Ginsenoside Rb1 was supplied by Hefei Bomei Biotechnology CO., LTD., China (cat. no. BZP0234); Rat antibody array 90 glass slide kit was supplied by RayBiotech, United States (cat. no. AAR-BLG-1); β-catenin antibody was purchased from Thermo Fisher Scientific, Inc., United States (cat. no. MA1-301); Transcription factor 7-like 2 (TCF-4/TCF7L2) antibody, c-myc antibody, cyclin D1 antibody, baculoviral IAP repeat containing 5 (Birc5) antibody and proliferating cell nuclear antigen (PCNA) antibody were purchased from abcam, United Kingdom ; Wnt1-inducible signaling pathway protein 1 (Wisp1) antibody, and c-jun antibody were obtained from Bioss, Inc., China . Ki67 antibody was supplied by Novus Biologicals, LLC, United States (cat. no. NB500-170). TdT-mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay kit was supplied by Boster Biological Technology CO., LTD., China (cat. no. MK1025).n = 10): 1) Normal ; 2) Model ; 3) Model + GRb1 . MNNG is a carcinogen, which can induce the occurrence of gastric precancerous lesions  once daily. At the end of 20th week, all animals were humanely anesthetized with sodium pentobarbital (140 mg/kg i. p.) after 12 h fasting. Following sacrifice by cervical dislocation, stomachs were harvested immediately. The flow chart of the experiment is shown in Animals were randomly assigned to three groups as follows  and then embedded in paraffin. To examine pathological morphology, paraffin-embedded samples were cut into 5 μm-thick sections and then stained with hematoxylin and eosin. Each slide was monitored under microscopy  and images were captured. The magnifications used were ×400. These slides were evaluated by two independent pathologists in a blinded manner.3 blocks and fixed in 2.5% glutaraldehyde in cacodylate buffer (pH 7.4) at 4°C for 4 h, and then post-fixed in 1% osmium tetroxide, also in phosphate buffer for 2 h. Next, samples were routinely dehydrated in graded ethanol series  and transferred to acetone solution for 20 min. For infiltration, the samples were immersed in a mixture of acetone and epoxy resin twice , and then embedded in epoxy resin-filled capsules. Finally, 70 nm ultrathin sections were counterstained with 2% aqueous uranyl acetate and 0.8% lead citrate. Ultrastructure of gastric epithelial cells was observed and photographed using a transmission electron microscope . The magnification was ×10,000.Fresh gastric mucosa was cut into 1 mmAB-PAS staining was performed to evaluate the severity of IM lesion. Neutral mucins present in normal mucosa were stained magenta, while acidic mucins present in IM lesion were stained blue. Then, HID-PAS staining was conducted to further assess small intestinal-type metaplasia (S-IM) and colonic-type metaplasia (C-IM), in which sulfomucins expressed only in C-IM lesion were stained brown. Images were captured under microscopy  with magnification of ×100.Biotin label-based rat antibody array was conducted to determine the expression levels of 90 rat proteins, according to the manufacturer’s instructions. Briefly, gastric epithelium was collected and homogenized in pre-cooled lysis buffer containing protease inhibitor cocktail at 4°C. Homogenates were then centrifuged at 12,000 rpm for 15 min twice. Next, supernatants were dialyzed in separate dialysis tubes, followed by determination of the total protein concentration using the bicinchoninic acid (BCA) assay . After biotin-labeling the samples and blocking process, 400 μl of diluted samples was added into each well for incubation overnight at 4°C. Then, each sub-array was incubated with Cy3-Conjugated Streptavidin at room temperature for 2 h (avoid exposure to light). Finally, the glass slides were scanned to detect the fluorescent signals of microarrays using an InnoScan 300 Microarray Scanner  at a wavelength of 532 nm.Paraffin-embedded gastric tissues were cut into 3 µm-thick sections, and dewaxed with xylene at room temperature and rehydrated in a descending ethanol series . For antigen retrieval, sections were heated at 97°C for 20 min. Following a peroxidase blocking with 3% hydrogen peroxide for 15 min and 5% bovine serum albumin blocking for 30 min, the sections were incubated with primary antibodies against β-catenin (1:500), TCF-4 , PCNA (1:500) and Ki67 (1:100) overnight at 4°C. The sections were then exposed to HRP/Fab polymer conjugate at room temperature for 30 min , following which they were stained with 3,3′-diaminobenzidine solution for 5 min and counterstained with hematoxylin for 20 s at room temperature. Three random visual fields were selected and photographed from each section. Quantification of expression levels was determined by mean of integrated optical density (IOD) using Image Pro Plus 6.0 software . Moreover, the expression pattern of β-catenin and its relationships with clinicopathological characteristics of GPL patients were evaluated. The sections were assessed by two independent investigators, without prior knowledge of the clinicopathological data, in a blinded manner. The immunoreactivity scores (IRS) were determined by the sum total of the percentage of positive cells , and staining intensity scores . A final IRS ≥4 indicated strong positivity, while scores <4 indicated weak positivity.TUNEL apoptosis assay was carried out according to the manufacturer’s instructions. Briefly, 3 µm-thick sections, sliced from paraffin-embedded gastric tissues, were routinely dewaxed and rehydrated. The sections were incubated with proteinase K working solution at 37°C for 15 min, followed by incubation with labeling buffer containing Terminal deoxynucleotidyl Transferase (TdT) and digoxin labeled deoxyuridine triphosphate (dUTP). Next, proceed with blocking reagent prior to incubation with biotinylated anti-digoxin antibody at 37°C for 30 min. Finally, samples were exposed to strept avidin-biotin complex (SABC), followed by staining with 3, 3′-diaminobenzidine solution and counterstaining with hematoxylin. Apoptotic cells were shown as nuclear staining. Counting for apoptotic cells was performed using light microscopy  with magnification of ×200. Three randomly selected fields were captured for each section, in which apoptosis index was calculated.Frozen gastric epithelium was collected, and homogenates and supernatants were prepared as described above. Total protein concentration was determined using the BCA assay . Then, 480 μg of total protein were incubated with 20 μl of protein A/G beads  at 4°C for 1 h (the negative controls were incubated with 20 μl of protein A/G beads and 2 μl lgG). After centrifugation, 2 μl β-catenin antibody (1:200) was added in lysate supernatants for incubation overnight at 4°C, and, next, 20 μl of protein A/G beads was mixed, followed by gentle rotation at 4°C for 2 h. Following centrifugation and removal of supernatants, the beads were collected. The immunoprecipitation complex was washed 4 times, mixed with reduced loading buffer, boiled, and centrifuged. Finally, western blotting was performed to detect the abundance of both β-catenin and TCF-4 proteins in the immunoprecipitation complex. To determine the protein expression levels of c-myc, cyclin D1, Wisp1 and Birc5, western blotting was also performed as described previously .c-myc, c-jun, cyclin D1, Wisp1 and Birc5 were determined using StepOne Plus real-time PCR System (Applied Biosystems Inc.). Thermal conditions were as follows: 10 min at 95°C, 40 cycles of 15 s at 95°C and 60 s at 60°C, with 0.3°C rise per 15 s from 60°C to 95°C. Gapdh was applied as an endogenous control for RNA input. Differences in amplification were calculated using the 2−△△Ct method. The primer sequences used were as follows: c-myc forward 5′-AAAACCCGACAGTCACGACG-3′ and reverse 5′-GTAGCGACCGCAACATAGGAC-3′; cyclin D1 forward 5′-TTCATCGAACACTTCCTCTCCA-3′ and reverse 5′-GAGGGTGGGTTGGAAA TGAA-3′; c-jun forward 5′-GCAATGGGCACATCACCACTAC-3′ and reverse 5′-GTGACACTGGGCAGCGTATTCT-3′; Wisp1 forward 5′-ACATCAAGGCAGG GAAGAAATG-3′ and reverse 5′-CCTCTGGACACTGGAAATCAAC-3′; Birc5 forward 5′-GACCACCGGATCTACACCTTC-3′ and reverse 5′-CTCGGTAGGGCA GTGGATGAA-3′; Gapdh forward 5′-CTGGAGAAACCTGCCAAGTATG-3′ and reverse 5′-GGTGGAAGAATGGGAGTTGCT-3′.Total RNA from gastric epithelium was extracted using a TRIzol kit . RNA concentration and quality were determined using the spectrophotometer  and 1% agarose gels. Complementary DNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit . Subsequently, mRNA levels of p value < 0.05, differential expression is considered. The other statistical data was analyzed using SPSS 23.0 software (IBM Inc.). Differences between groups were evaluated using one-way analysis of variance, followed by Tukey method for homogeneous data and Dunnett’s T3 method for non-homogeneous data. Data are expressed as the mean ± standard deviation. p < 0.05 was considered to indicate a statistically significant difference. Unpaired Student’s t-test was used to assess the differences in β-catenin expression levels between gastric precancerous lesions group and control group. Pearson’s χ2 test and Fisher’s exact test were used to assess the association between nuclear localization of β-catenin, β-catenin expression and clinicopathological characteristics.For antibody array assay, R software (version 3.6.3) package “limma” was employed to analyze the differential expression proteins using a linear model of empirical Bayesian method. When We test the effects of GRb1 on pathomorphology of gastric epithelium in GPL rats on macroscopic, microscopic and ultramicroscopic levels. As shown in We then examined the degree of IM lesion in gastric epithelium using AB-PAS staining. Neutral mucins present in normal gastric epithelium were stained magenta, while acidic mucins present in IM lesion were stained blue . As depicted in To screen differential expression proteins (DEPs) in the GPL rats relative to normal controls, we carried out antibody array assay. Statistical analysis results unveiled that, compared to the normal group, seven DEPs were screened out in the GPL model group . Among tIn order to verify the role of β-catenin in precursors of gastric cancer, we investigate the expression and subcellular localization of β-catenin in 94 cases of human GPL specimens and in 85 cases of gastric specimens from the healthy. β-catenin expression was identified in 80.9% (76/94) of the GPL specimens and 72.9% (62/85) of the normal specimens. The β-catenin immunoreactivity was notably stronger in the human GPL specimens than in the healthy controls . In the Association between clinical characteristics and β-catenin positivity/subcellular localization in patients with GPL was investigated. Our analysis demonstrated that rate of nuclear localization of β-catenin was significantly different between normal gastric epithelium and GPL samples. Moreover, nuclear expression of β-catenin in gastric epithelium increased depending on the pathological grade of GPL . In the GPL specimens assessed, high percentage of nuclear staining with β-catenin was not significantly associated with age, sex and location of lesion . AnalysiOur verification results in the present animal study showed that β-catenin protein expression was increased and β-catenin preferentially accumulated in the cytoplasm and, in some cases, translocated into the nucleus in GPL model rats . In accoTo further confirm the effects of GRb1 on the downstream target genes of β-catenin, such as c-myc, cyclin D1, c-jun, Wisp1 and Birc5 that are closely associated with cellular proliferation and apoptosis, RT-PCR was employed to define the transcriptional levels of these target genes. The protein levels of these target genes were determined by Western blot. It appeared that mRNA and protein levels of the genes detected were significantly higher in the GPL rats than in the normal controls. When compared with the GPL rats, mRNA levels of c-myc, cyclin D1 and Birc5 were significantly down-regulated in the GRb1-treated GPL rats , and proThe proliferation and apoptosis of gastric epithelial cells was further evaluated. Firstly, the protein expression of PCNA and Ki-67, which are widely considered as the specific reporters for cell proliferation, were determined. Our IHC analysis demonstrated that the expression of PCNA and Ki-67 was significantly higher in GPL model rats than in the normal controls, suggesting that GPL rats were experiencing hyper-proliferative state of epithelial cells relative to normal rats; while GPL rats treated with GRb1 achieved a significant decrease in the expression of PCNA and Ki-67 proteins . We thenIn the present study, we found that GRb1 administration reverse intestinal metaplasia and a portion of dysplasia in the MNNG-induced GPL rats. To screen targeting DEPs and investigate the possible mechanism, we applied antibody array assay and screened out seven DEPs in GPL model group relative to normal group, in which three DEPs  were significantly down-regulated after GRb1 administration. Among the three DEPs, β-catenin was the therapeutic target we were most interested in, because our earlier study showed that an herbal formula Weipixiao, of which GRb1 is the major bioactive constituent , could aβ-catenin is an integral structural component of cell adherens junctions and a key downstream effector of canonical Wnt pathway . It has It has been recognized that Wnt/β-catenin/TCF-4 pathway is often aberrantly activated in gastric cancer , in whicPreviously, increased transcription levels of c-myc, cyclin D1, c-jun, Wisp1 and Birc5 in the development and progression of gastric cancer were reported . As expeIn summary, we showed the novel finding that GRb1 prevented the occurrence and progression of gastric precancerous lesions. The therapeutic effects might be contributed by decreasing protein expression and nuclear translocation of β-catenin and interfering with β-catenin/TCF4 interaction, and by repressing the transcriptional activity of downstream genes including c-myc, cyclin D1 and Birc5.


Wnt/β-catenin stimulation instructed the neuronal differentiation of proliferating NSCs and promoted the activation or differentiation of quiescent NSCs in a dose-dependent manner. However, deletion of β-catenin in NSCs did not affect either their activation or maintenance of their stem cell characteristics. Together, these results indicate that, although NSCs do respond to Wnt/β-catenin stimulation in a dose-dependent and state-specific manner, Wnt/β-catenin signalling is not cell-autonomously required to maintain NSC homeostasis, which reconciles some of the contradictions in the literature as to the role of Wnt/β-catenin signalling in adult hippocampal NSCs.Adult mouse hippocampal neural stem cells (NSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood and memory. These NSCs are largely quiescent and are stimulated by niche signals to activate and produce neurons. Wnt/β-catenin signalling acts at different steps along the hippocampal neurogenic lineage, but whether it has a direct role in the regulation of NSCs remains unclear. Here, we used Wnt/β-catenin reporters and transcriptomic data from  Summary: Wnt/β-catenin signalling stimulation promotes the exit from quiescence and differentiation of adult hippocampal neural stem cells but is dispensable for homeostatic neurogenesis in the dentate gyrus of young mice. Adult neurogenesis, the continuous process of generating new neurons from neural progenitors throughout life, is predominately restricted to two adult brain regions in mice: the ventricular-subventricular zone (V-SVZ) of the lateral ventricles and the dentate gyrus (DG) of the hippocampus, in which adult-born neurons contribute to olfactory bulb and hippocampal functions, respectively . Adult hAxin2  and Wnt inhibitors (Dkk3 and Sfrp1), suggesting that NSCs respond to, and directly regulate, Wnt activity levels in the DG  at a higher level compared with active NSCs, which corroborates previous reports showing that quiescent NSCs are enriched for cell surface receptors  and GSK3β (Gsk3b) are upregulated in active NSCs (Axin2 was expressed at low levels by very few quiescent (5%) and active (7%) NSCs (Quiescent NSCs expressed Wnt receptors (eceptors A (Shin eeceptors . The Wntive NSCs A. Howeve7%) NSCs A, which 7%) NSCs .Axin2 levels between quiescent and active NSCs suggest that cells in these two states respond similarly to Wnt/β-catenin signalling  Wnt/β-catenin reporter mice, which express β-galactosidase (Glb1) under the control of 7xTCF/LEF-binding sites ; therefore, we generated a second β-catenin floxed mouse line: fl/fl ex2-6; RYFPGlastCreERT2; β-cat mice  (Ctnnb1 transcript was significantly decreased in YFP-positive fluorescence-activated cell (FAC)-sorted cells from the DG of β-catdel ex2-6 mice compared with controls , indicating the successful recombination of the fl/fl ex2-6β-cat allele. β-catenin protein levels were low in the DG, especially when compared with the SVZ . Nevertheless, we were able to detect β-catenin staining in the radial processes of NSCs in control mice, which was eliminated in recombined (YFP-positive) NSCs , confirming the deletion of β-catenin protein. However, Axin2 transcript levels were very low and unchanged between YFP-positive FAC-sorted cells from the DG of β-catdel ex2-6 mice compared with controls . Given that beta-galactosidase accumulates in the cell, it can be a more-sensitive readout of Wnt activity. Therefore, we crossed β-catdel ex2-6 mice with BATGAL mice and found that the proportion of BATGAL-positive NSCs was reduced in β-catdel ex2-6 mice compared with controls, confirming that the loss of β-catenin impairs Wnt/β-catenin activity in NSCs . To examine the effect of β-catenin deletion on NSCs, we administered tamoxifen to 2-month-old β-catdel ex2-6 and control mice and performed immunofluorescence analysis 30 days later, focusing on recombined YFP+ cells  after tamoxifen administration and found no significant difference between genotypes, indicating that NSC maintenance was unaffected by β-catenin deletion. Overall, these data suggest that the proliferation and maintenance of NSCs are unaffected by loss of Wnt/β-catenin signalling.To investigate the effects of Wnt/β-catenin inhibition in NSCs bination . Howeverig. S2A) . The expP+ cells A,B. The feration C. We alsferation D and 90 del ex2-6 and control mice 30 days after β-catenin deletion. We also quantified the total number of neuroblasts and newly generated neurons 90 days after tamoxifen administration and again observed no difference between genotypes . Overall, these data show that loss of Wnt/β-catenin signalling in NSCs in vivo does not impair the behaviour or maintenance of NSCs; neither does it impair the generation and survival of new neurons in the adult hippocampus.We then investigated how the loss of β-catenin affects later steps in adult hippocampal neurogenesis. We quantified the percentage of proliferating IPCs E, the toin vivo, we investigated how stimulating Wnt/β-catenin signalling would affect NSCs. To do so, we generated lox(ex3)/wt RYFPGlastCreERT2; Catnb mice [hereafter referred to as Catnbdel(ex3) mice] to stabilise β-catenin conditionally in Glast-expressing NSCs (lox(ex3)Catnb is a conditional constitutively active allele of β-catenin (Catnb) in which exon 3, which encodes the GSK3β phosphorylation sites that mark β-catenin for degradation, is flanked by LoxP sites (lox(ex3)Catnb allele resulted in β-catenin stabilisation and ligand-independent activation of downstream Wnt/β-catenin signalling in targeted cells. Upon recombination, we observed an increase in both β-catenin levels and in the intensity of the BATGAL reporter in lox(ex3)Catnb/BATGAL mice compared with BATGAL controls .After having assessed the effects of disrupting Wnt/β-catenin signalling in NSCs ing NSCs . CatnbloxP sites . Cre-meddel(ex3) and control mice with tamoxifen for 5 consecutive days and analysed their NSCs 10 and 30 days later . We first assessed the effect of stabilising β-catenin in NSCs by quantifying the total number of NSCs in Catnbdel(ex3) and control mice . We found a significant decrease in the number of NSCs in Catnbdel(ex3) mice compared with controls at 30 days  but not at 10 days after tamoxifen administration , suggesting that stabilising β-catenin causes NSC loss between 10 and 30 days later. This could be the result of increased NSC proliferation and subsequent depletion. We observed an increase in the proportion of Ki67-positive NSCs 30 days but not 10 days after tamoxifen administration . Therefore, the loss of NSCs at 30 days could be because of increased proliferation between the 10 and 30 day time points. However, we noticed that the cellular organisation of the DG was already disrupted in Catnbdel(ex3) mice 10 days after β-catenin stabilisation. Many YFP-positive recombined cells were displaced throughout the GCL and molecular layer (ML) . Some of these cells retained NSC characteristics  but their cell bodies were not correctly located in the SGZ . Such cells were also present in control mice, but the proportion of displaced NSCs was increased three-fold in Catnbdel(ex3) compared with control mice . This suggests that stabilising β-catenin in NSCs promotes their displacement from their correct SGZ location, which might cause their subsequent loss from the DG. Given that β-catenin regulates cell adhesion at adherens junctions . We next examined Axin2 levels in β-catdel ex3-6 and control NSCs treated with CHIR99021, which stimulates Wnt/β-catenin signalling by inhibiting GSK3β . Of note, the basal levels of Axin2 did not change upon β-catenin deletion, indicating that Wnt activity is very low in proliferating NSCs in vitro as it is in vivo.We next took advantage of this YFP mice , hereaftng GSK3β . CHIR990del ex3-6 active NSCs were pulsed with EdU for 1 h to label cells in S-phase of the cell cycle at different time points after Cre transduction  and Sox2 were similarly expressed between β-catdel ex3-6 and control active NSCs at all time points  remains unaddressed. To investigate this, we returned β-cattivation I. We meativation I,J. Prolve cells K. We alsnduction L. This sdel ex3-6 and control active NSCs at 6-, 12- and 18-days after Cre transduction to promote neuronal differentiation (B27 for 5 days) or astrocytic differentiation (FBS for 5 days) . We then immunolabelled cells for the neuronal marker MAP2 and astrocyte marker GFAP to identify and quantify the proportions of neurons and astrocytes, respectively . We found that β-catdel ex3-6 NSCs generated a similar proportion of MAP2-positive neurons to control NSCs . The proportions of GFAP-positive astrocytes generated were also similar between β-catdel ex3-6 and control NSCs . This suggests that the neurogenic and gliogenic potential of NSCs is unaffected by the loss of β-catenin-dependent Wnt signalling under these differentiating conditions in vitro.Given that NSC proliferation and activity were not affected by loss of Wnt/β-catenin signalling, we next tested whether neuronal or astrocytic differentiation was affected by the chronic loss of β-catenin. We changed the media conditions of β-catAxin2 expression levels, which were significantly higher compared with control cells in response to treatment with 5 µM and 10 µM CHIR99021 ]. We found that the expression of Tuj1 as well as the proportion of TUJ1-positive immunolabelled cells were increased in active NSCs upon 10 µM CHIR99021 treatment  did not reduce the already very low levels of proliferation of quiescent NSCs B,C. Howeve cells D-F, whicve cells D-F. Remave cells B,C. Thisve cells B,C. We aol cells D-F. OverAxin2 expression levels across all time points , indicating a constant level of Wnt/β-catenin stimulation over time. The proportion of Ki67-positive cells increased with time in 5 µM CHIR99021-treated quiescent NSCs , whereas the expression of Tuj1 was not significantly upregulated at any time point . Overall, this suggests that sustained 5 µM CHIR99021 treatment promotes the activation of an increasing proportion of quiescent NSCs but does not initiate their neuronal differentiation.We next investigated whether the duration of CHIR99021 treatment influences the response of quiescent NSCs. To do so, we performed a time course study, in which quiescent NSCs were treated with 5 µM CHIR99021 for 24 h, 48 h and 72 h. We found that 5 µM CHIR99021 treatment induced a similar increase in Axin2 was upregulated in a dose-dependent manner by different concentrations of rWnt3a, with 500 ng/ml rWnt3a inducing Axin2 expression to a similar level as 5 µM CHIR99021 . To further test the capability of β-catenin to promote NSC activation, we generated a NSC line from Catnblox(ex3) mice, induced quiescence and then stabilised β-catenin by the addition of Cre virus (or Null virus as control) . β-catenin was effectively stabilised following Cre transduction of Catnblox(ex3) NSCs, and the number of proliferating cells was higher in Catnbdel(ex3) NSCs compared with controls . In addition, there was a slight increase in the proportion of TUJ1-positive cells , reminiscent of the increase in TUJ1-positive cells induced by 5 µM CHIR99021 treatment (We confirmed the results obtained with CHIR99021 by treating quiescent NSCs with recombinant Wnt3a (rWnt3a). HIR99021 A. rWnt3aHIR99021 B-D. Addireatment F, suggesin vivo and in vitro. We found that Wnt/β-catenin signalling was low in the DG niche and that, whereas Wnt/β-catenin inhibition did not affect NSC homeostasis, Wnt/β-catenin stimulation exerted dose-dependent and state-specific effects on NSCs.In this study, we used genetic and pharmacological approaches to stimulate and inhibit Wnt/β-catenin signalling directly in quiescent and active NSCs, both in vitro or in vivo. Our finding that baseline Wnt/β-catenin signalling was not essential for the maintenance of the activation states or stem cell characteristics of NSCs differs from published reports showing that ablation of endogenous Wnt/β-catenin signalling affects NSC proliferation and multipotency  mice. Disruption of β-catenin binding to actin via α-catenin could affect the adhesion properties of recombined NSCs, contributing to the displacement phenotype we observed and to their subsequent loss. Indeed, disruption of β-catenin/cadherin signalling and loss of cell-cell contacts can disrupt tissue organisation and promote cell migration (del ex2-6 mice (del(ex3) mice prevented us from using this mouse model to investigate the effects of Wnt/β-catenin stimulation on NSC behaviour. To overcome this problem, we used an in vitro model of hippocampal NSCs, which allows the culture of pure and homogeneous populations of NSCs in a controlled environment. We showed that stimulating Wnt/β-catenin signalling promotes neuronal differentiation of active NSCs while promoting the proliferation or differentiation of quiescent NSCs in a dose-dependent manner. The dose-dependent effects of stimulating Wnt/β-catenin signalling in quiescent NSCs shown here could provide a possible explanation for some of the contradictions reported in the literature, because different techniques used to modulate Wnt could stimulate Wnt/β-catenin signalling levels to varying degrees MgoeSlc1a3] (Rosa26-floxed-stop-YFP  (fl/fl ex3-6 (tm2BirCtnnb1) (fl/fl ex2-6 (tm2KemCtnnb1) (lox(ex3) (tm1MmtCtnnb1) ; RYFPGlast-CreERT2; Catnb mice were crossed with BATGAL Wnt/β-catenin reporter mice . Mice were housed in standard cages under a 12 h light/dark cycle with T2)Mgoe]  mice werYFP)Cos]  mice to 1tm2Bir) , β-catfl1tm2Kem)  and Catn1tm1Mmt) . Glast-Cter mice  to introin vivo studies. Ages of animals used are detailed below or in the figure legends.Experimental groups included a combination of mice from different litters within each strain, and both males and females were used for all 2 and routinely tested for mycoplasma contamination.Adult hippocampal NSC cultures were derived as previously described . Brieflyfl/fl ex3-6; RYFPGlastCreERT2; β-cat mice were administered 2 mg (57-67 mg/kg) 4-hydroxy-tamoxifen  by intraperitoneal injection at the same time each day for 5 consecutive days. Given the high numbers of deaths, and following veterinary advice, we changed to use tamoxifen  for all other experiments. Two-month-old mice were administered 2 mg (57-67 mg/kg) tamoxifen by intraperitoneal (ip) injection at the same time each day for 5 consecutive days.To induce activation of CreERT2 recombinase, 2-month-old lox(ex3); RYFPGlast-CreERT2; Catnb mice developed a skin phenotype resulting from the stabilisation of β-catenin in Glast-expressing activated hair follicle stem and progenitor cells  in PBS for 12 min. Brains were removed and post-fixed overnight in 4% PFA at 4°C with rocking and then transferred to PBS containing 0.02% sodium azide. Brains were coronally sectioned at a thickness of 40 µm using a vibratome (Leica).2O for 30 min and fixed NSCs were incubated with 2 µg/ml DAPI in 1:1 PBS:H2O for 10 min at room temperature. Primary and secondary antibodies and dilutions are listed in Table S1. EdU was detected prior to DAPI incubation using the Click-iT™ EdU Alexa Fluor 647 detection kit  following manufacturer's instructions.Cultured NSCs were fixed with 4% PFA in PBS for 10 min at room temperature and then washed with PBS. Antigen retrieval was performed in brain sections prior to immunofluorescence using the following antibodies: GFP , β-catenin , DCX , Ki67 , SOX2  and TBR2 . For antigen retrieval, samples were incubated at 95°C for 2 min in sodium citrate buffer . Following antigen retrieval, samples were processed for immunofluorescence as previously described . Prior tz-step. For each brain section, the left and right DGs in every 12th 40 µm section along the rostrocaudal axis were imaged, with a 1 µm z-step through the whole 40 µm section. For the reactivation experiment in quiescent control and β-catdel ex3-6 NSCs and quiescent Catnblox(ex3)/wt NSCs, an Axio Imager.Z2 microscope with a 20× objective (Zeiss) with Hamamatsu Orca Flash 4 camera and Apotome 2 technology for optical sectioning was used. Images of the comparison between DG and SVZ for immunolabelling of β-catenin were acquired with an Axio Observer microscope (Zeiss) equipped with a Yokogawa CSU X1 spinning disk and a Hamamatsu EMCCD camera.Immunofluorescence samples were imaged using a SP5 confocal microscope (Leica) with a 40× oil objective lens (Leica). For fixed NSCs, three random regions of each coverslip were imaged with a 1 µm 2 to establish quiescence as previously described  and incubated for 72 h at 37°C, 5% COescribed . All expTo label cells in S-phase of the cell cycle, EdU  was added to the media of cultured cells at a final concentration of 10 µM 1 h before fixation.fl/fl ex3-6; RYFPβ-cat mice, active and quiescent β-catfl/fl ex3-6 NSCs were transduced with either adenovirus control  or adenovirus expressing Cre  at a multiplicity of infection (MOI) of 100. Samples of active β-catfl/fl ex3-6 NSCs were collected 1, 2 and 3 days after transduction for quantitative (q)PCR, immunofluorescence and western blot analyses. Samples of quiescent β-catfl/fl ex3-6 NSCs were collected 2, 4 and 6 days after transduction for qPCR and immunofluorescence analyses. To stimulate Wnt/β-catenin signalling in active β-catfl/fl ex3-6 NSCs, media was replaced with basal media plus 5 µM CHIR99021  or DMSO vehicle control 48 h after transduction and incubated for 24 h at 37°C, 5% CO2 before collection in TRIzol  for RNA extraction, cDNA synthesis and qPCR analysis. To determine the reactivation rate of quiescent β-catfl/fl ex3-6 NSCs, media was replaced with basal media 48 h after transduction and samples were collected 3 and 6 days later for immunofluorescence analysis. Samples were also collected in basal media plus 20 ng/ml BMP4 48 h after transduction for the 0 h time point. Media was refreshed every 72 h. To determine how chronic loss of β-catenin affects neurogenesis and gliogenesis, the media on transduced active β-catfl/fl ex3-6 NSCs was replaced at 6, 12 and 18 days after transduction with basal media minus 20 ng/ml FGF2 plus 2% B27 to promote neuronal differentiation and basal media minus 20 ng/ml FGF2 plus 2% FBS to promote astrocytic differentiation. Cells were incubated in these differentiation media conditions for 5 days at 37°C, 5% CO2 before fixation. To confirm that 5 µM CHIR99021-induced reactivation of quiescent NSCs was dependent upon Wnt/β-catenin signalling, quiescent control and β-catdel ex3-6 NSCs were treated with either DMSO vehicle control or 5 µM CHIR99021 for 48 h before fixation for immunofluorescence analysis. Of note, we observed that treatment with CHIR99021 immediately after viral transduction often resulted in poor viability of the cultures. Therefore, we performed this experiment using two alternative protocols, with the viral transduction step either in proliferating or already-quiescent NSCs .To delete β-catenin in NSCs derived from 2 before sample collection. To stimulate Wnt/β-catenin signalling in quiescent NSCs, media on cells was replaced with basal media plus 20 ng/ml BMP4 supplemented with DMSO vehicle control, 1 µM CHIR99021, 5 µM CHIR99021 or 10 µM CHIR99021 and incubated for 24 h, 48 h and 72 h at 37°C, 5% CO2 before sample collection. To compare the effects of CHIR99021 treatment versus rWnt3a  in quiescent NSCs, media on cells was replaced with basal media plus 20 ng/ml BMP4 supplemented with DMSO (vehicle control for CHIR99021), 5 µM CHIR99021, 0.1% BSA/PBS (vehicle control for rWnt3a), 100 ng/ml rWnt3a, 250 ng/ml rWnt3a or 500 ng/ml rWnt3a and incubated 48 h at 37°C, 5% CO2 before sample collection. Media was refreshed every 24 h. To stabilise β-catenin in NSCs derived from lox(ex3); RYFPCatnb mice, quiescent Catnblox(ex3)/wt NSCs were transduced with either Ad-CMV-Null or Ad-CMV-iCre at a MOI=100. Control and Catnbdel(ex3) NSCs were collected 2 days after virus transduction for immunofluorescence analysis.To stimulate Wnt/β-catenin signalling in active NSCs, media on cells was replaced with basal media supplemented with DMSO vehicle control, 1 µM CHIR99021, 5 µM CHIR99021 or 10 µM CHIR99021 and incubated for 48 h at 37°C, 5% COg) at 4°C for 20 min. The supernatant was transferred to a clean Eppendorf tube and an equal volume of Laemmli sample buffer  was added before boiling at 95°C for 5 min.For western blot analysis, samples were washed with ice-cold PBS and scraped in lysis buffer  supplemented with a phosphatase inhibitor , EDTA  and a protease inhibitor . Cells were lysed at 4°C for 20 min under rotation and then centrifuged at 13,000 rpm  or 5% BSA in TBS-Tween before incubation with primary antibody diluted in 5% milk in TBS-Tween  or 5% BSA in TBS-Tween+0.02% sodium azide (Wnt7a) overnight at 4°C under rotation. The following day, membranes were washed with TBS-Tween and incubated with secondary antibody diluted in 5% milk in TBS-Tween for 2 h at room temperature with rocking. Detection was performed using ECL Western Blotting reagents  according to the manufacturer's instructions and blots were developed using Kodak films. Primary and secondary antibodies and dilutions are listed in del ex3-6 and β-catdel ex2-6 mice were injected with tamoxifen for 5 consecutive days and culled 10 days later. The DG was dissected as previously described  for RNA extraction.Two-month-old control, β-catescribed . Dissectescribed . DissociTable S2) and qPCR was performed on a QuantStudio Real-Time PCR machine (Thermo Fisher Scientific). Gene expression was calculated relative to the endogenous controls GAPDH and ActinB (dCt) and to the control samples to provide a delta delta cycle threshold (ddCt) value. ddCT values were then converted to the fold change in gene expression relative to control. All samples were measured in technical duplicates for each qPCR run and then averaged.For FACS experiments, cells were lysed using the RL lysis buffer from the RNeasy Mini Kit. For all other experiments, cells were lysed using TRIzol reagent . RNA was extracted using the RNeasy Mini Kit or the Direct-zol RNA MiniPrep Kit  according to the manufacturers’ instructions. cDNA was synthesised using the Maxima First Strand cDNA Synthesis Kit  or the High Capacity cDNA Reverse Transcription Kit  according to the manufacturers’ instructions. Gene expression levels were measured using TaqMan Gene expression assays . The final dataset contained 2947 NSCs, which were classified as dormant, resting and active NSCs as described by t-test  were made using a ring mask around each nucleus. The proportion of cultured NSCs expressing specific markers was quantified by setting a signal threshold and expressing the number of positive cells as a percentage of the total number of cells. For each experiment, at least 100 cells were quantified across three biological replicates unless stated otherwise.n) was based on experiments from our previous publications  or GraphPad Prism 9 software (Version 9.2.0. RRID:SCR_002798). Appropriate statistical tests for each type of data were used as follows: a two-tailed unpaired Student's


Wnt signaling plays a critical role during embryogenesis and is responsible for regulating the homeostasis of the adult stem cells and cells fate via a multitude of signaling pathways and associated transcription factors, receptors, effectors, and inhibitors. For this review, published articles were searched from PubMed Central, Embase, Medline, and Google Scholar. The search terms were Wnt, canonical, noncanonical, signaling pathway, β-catenin, environment, and heavy metals. Published articles on Wnt signaling pathways and heavy metals as contributing factors for causing diseases via influencing Wnt signaling pathways were included. Wnt canonical or noncanonical signaling pathways are the key regulators of stem cell homeostasis that control many mechanisms. There is an adequate balance between β-catenin dependent and independent Wnt signaling pathways and remain highly conserved throughout different development stages. Environmental heavy metal exposure may cause either inhibition or overexpression of any component of Wnt signaling pathways such as Wnt protein, transcription factors, receptors, ligands, or transducers to impede normal cellular function via negatively affecting Wnt signaling pathways. Environmental exposure to heavy metals potentially contributes to diseases via deregulated Wnt signaling pathways.  Wnt signaling plays a critical role during embryogenesis. It is responsible for regulating the homeostasis of the adult stem cells and cells fate via a multitude of signaling pathways and associated transcription factors, receptors, effectors, and inhibitors (Wnt is derived from the combination of two gene names of Drosophila melanogaster wingless (wg1) and mouse proto-oncogene (int-1)  and noncanonical (β-catenin independent) pathways. In the same way, Wnt proteins are classified into canonical Wnts  and noncanonical Wnts   . After ts ‘wint’ , 5. So fCanonical Wnt signaling pathwaysWnt signaling or Wnt/β-catenin signaling regulates cell fate determination during embryonic development. An absence of Wnt protein extracellularly results in activation of β-catenin destruction complex (βCDC), which consists of two scaffold proteins, i.e., axin and adenomatous polyposis coli (APC) and two kinases enzymes, i.e., casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3) that are responsible for the phosphorylation of β-catenin. Phosphorylated β-catenin is recognized by F-box protein β-transduction repeat-containing protein (β-Trcp), which along with Skp1-Cullin-F-box (SCF) ubiquitin ligase, target β-catenin for proteasomal degradation and prevents its translocation to the nucleus. In the absence of β-catenin, Groucho/TLE binds to T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factor recruiting histone deacetylase (HDAC) and thereby repressing transcription of many downstream genes (Wnt protein forms a Wnt-Fz-LRP6/5 complex upon binding to frizzled (fz) receptor and its co-receptors LRP6/5. The Wnt-Fz-LRP6/5 complex recruit cytosolic protein disheveled (Dv1), engaging Gsk3 binding protein (GBP) and axin along kinases enzymes CK1 and Gsk3, which in turn phosphorylate LRP6/5. The phosphorylated LRP6/5 acts as positive feedback causing sequestration of βCDC, consequently stabilizing and accumulating cytoplasmic β-catenin to enter into the nucleus and finally activating Wnt target gene expression  into the nucleus and transcribe downstream regulatory genes , axin, and Gsk3 to phosphorylate Ror1/2. Phosphorylated Ror1/2 activates phospholipase C on the plasma membrane, causing intracellular signaling activation and formation of inositol 1,4,5 triphosphate (IP3). IP3 and cytoplasmic diacylglycerol (DAG) diffuse to stimulate the endoplasmic reticulum and release of Ca2+ that finally leads to the activation and translocation of transcription factors into the nucleus and change in the expression of downstream regulatory genes (Wnt/planar cell polarity pathway initiated upon recruitment of Dvl by Fz and its co-receptors Ror/Ryk/PTK7 leading to actin cytoskeleton rearrangements. The MAP kinase (MAPK) and C-jun-N-terminal kinase (JNK) pathways activate C-Jun and API transcription factors . In . In Wnt ry genes . In the  factors  11-13).-13.Wnt sWnt antagonists Wnt antagonists can negatively affect Wnt signaling pathway activation. They can be classified based on their different mechanisms of antagonizing ligand-receptor interaction. Members of secreted frizzled-related proteins (sFRPs), Wnt inhibitory factor 1 (Wif-1), Cerberus (Cer), notum SOST, Wise, and angiopoietin-like 4 (ANGPTL4) are Wnt antagonists that bind to Wnt ligand and block Wnt signaling pathways. Unlike other Wnt antagonists, dickkopf (Dkk) family members inhibit Wnt signaling by binding to Wnt receptors  exposure induces malignant transformation  expression, an angiogenic stimulating growth factor augments β-catenin activity that leads to angiogenesis and risk of carcinogenesis (As(III)) or hexavalent chromium promoted colorectal tumor in azoxymethane/dextran sodium sulfate treated mice. As was found to induce tumorigenesis due to the ROS-mediated Wnt/β-catenin signaling pathway. As. by generating ROS caused imbalance of oxidant and antioxidant enzymes along with declined superoxide dismutase (SOD) and catalase level, while increased expression of β-catenin, phospho-GSK, NADPH oxidase1 (NOX1), and 8-OHdG. Suggesting the role of As. exposure to the tumor size increase, incidence, and inflammation via modulating the Wnt/β-catenin signaling pathway   expresses in adipocytes, whose inhibition impairs adipogenesis (ASCs) differentiation in mice vide As induced altered canonical TGFβ signaling pathway and dose-dependent decline in the β-catenin (CTNNB1), osteogenic such as Runx2, OPN, and BGP along with and chondrogenic such as Sox9, DSPG3 and ACAN gene expression , Cox2, MMP2 and MMP9 expression in RIMM-18 cells  . As expoogenesis . Persistcer risk . Environogenesis . Since Pogenesis , 29. Morogenesis , 31. Anopression . As. expignaling . Moreove by Wnt4 . BMP7 prl injury . Likewiselopment . Howeverigenesis , 38. SugCadmiumCd) as a carcinogen due to its toxic effect via releasing ROS, impairing calmodulin activity, and potential of altering signal transduction networks including Wnt/β-catenin, and estrogen  and progenitor cells towards myelopoiesis  via abnormal Wnt/β-catenin signaling and aryl hydrocarbon receptor targets genes including Ahr, Arnt, Nkx2.5, Ctnnb1 and Gsk3β. Thus impairs the physiological function of Ahr in regulating Wnt/β-catenin signaling that leads to the risk of CVDs  defect (Rho-associated coiled-coil-containing protein kinase (ROCK) I and ROCK-II regulates signaling from Rho to the actin cytoskeleton in Wnt non-canonical signaling pathway while it absence demonstrated ventral body wall (VBW) defect. A study on chick embryo demonstrated Cd induced downregulated ROCK I and ROCK-II genes expression during embryogenesis that resulted into VBW defect in chick embryo due disrupted Wnt non canonical signaling pathway  in the Wnt/Ca2+ pathway requiring for actin-cytoskeleton organization and cell adhesion , leading to renal fibrosis and the risk of developing cancer. Cd exposure considerably increases kidney Cd content which in turn increases expression of various Wnt ligands, including -3a,6,7a/b,9a/b,10a and 11 and upregulation of Fz1 to Fz10 except Fz3 receptor. Thus caused increased expression of Wnt target genes such as Abcd1b, c-Myc and cyclin D1 which promote cell proliferation, survival, migration and malignancy that leads to characteristic changes in the renal epithelial cells towards fibrosis and cancer through activated Wnt signaling pathway  defect . Rho-assadhesion , 53. Cd halocele . Cd repopression . Thymocyelopment . Cd expotoxicity . Chronic pathway . These fD1 genes . ChromiumEMT, metastasis, and Wnt signaling. There also observed upregulation of potential lung cancer biomarker ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) that initiates the transformation of lung epithelial cells towards an early stage of lung cancer  on BEAS-2B human lung epithelial cells demonstrated changes in the various gene expression mostly related to cell adhesion, protein ubiquitination, oxidative stress, g cancer . Another pathway . CopperCu) inhibits zebrafish egg hatching via suppressing embryonic motility  was found to alleviate the suppressing effect of Cu on egg hatching and swimbladder development  is an essential component of thick myofilament assembly while, its expression inhibits the cardiac looping resulting in impaired cardiac development  exposure Pb induces neurotoxic and extra neurotoxic pathophysiological outcome that tends to sustain and maintain for a lifetime (Pb exposure through lactation among rat pups demonstrated impaired learning and memory (activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) and hippocampal Wnt7a is known to regulate dendritic spines’ formation and structure  and sclerostin while decreases β-catenin and Runx2 in stromal precursor cells, thereby disrupt bone homeostasis via inhibition of the Wnt/β-catenin pathway , type 1 collagen (COL1), osteocalcin (OC), and Runx2 impairing regulation of Wnt3a, Dkk-1, pGSK3β, and β-catenin  and TGFβ, thus induce chondrogenesis  induces liver toxicity employing several processes associated with oxidative stress-mediated cell death, dysregulation of kinases including Gsk3 during Wnt signaling pathways. This gluconeogenesis and adipogenesis resulted in mitochondrial dysfunction, metabolic disruption, and endocrine disruption (Mercury (sruption . Wnt signaling pathways are vital for normal cellular functions and are sensitive to environmental exposure of heavy metals such as As, Cd, Cu, Pb, and Hg. Heavy metal exposure deregulates the Wnt signaling pathway that ultimately contributes to the initiation of various diseases and even cancer. Heavy metal-induced deregulated Wnt signaling pathway contributes to cancer and tumor development, toxicity to system organs such as kidney and liver, impairs normal bone and skeleton growth, and contributes toxicity to marine life. However, more research is warranted involving humans and exposure to other heavy metals to rule out their exact mechanism of action and possible means of controlling them to save humans, animals, and marine life. All authors contributed equally to this review.


TIB‐71 and RAW264.7CRL‐2278 cell lineages for their osteoclastic differentiation, and they concluded that the RAW264.7CRL‐2278 demonstrated to generate much osteoclast than RAW264.7TIB‐71. However, on the contrary to their results we noticed the fusion of RAW264.7TIB‐71 in our previous studies was much compromising. Therefore, we try to explore the two cell lineages for their properties in osteoclastic differentiation with an in‐depth cellular cytoskeletal study. Our current study has showed that comparing to the RAW264.7CRL‐2278, RAW264.7TIB‐71 demonstrated a much higher efficacies for RANKL‐stimulated osteoclastic differentiation. Besides that, in our depth cytoskeletal studies, we found that the RANKL‐induced RAW264.7TIB‐71 cells could finally differentiate into mature osteoclasts. However, regardless the various pre‐treatment conditions, there was no mature osteoclast formed in RANKL‐induced RAW264.7CRL‐2278 cell lineage.Although document studies (including ours) have been reported the achieved in vitro osteoclastic cellular model establishment from the RAW264.7 cell lineage, there was no study directly reported that American Type Culture Collection (ATCC) cell bank has various RAW264.7 cell lineages. Besides that, for our knowledge there was only one study compared the two different RAW264.7 However, on the contrary to their results we noticed the fusion of RAW264.7TIB‐71 in our previous studies was much compromising; therefore, we try to explore the two cell lineages for their properties in osteoclastic differentiation with an in‐depth cellular cytoskeletal study.Besides that, for our knowledge there was only one study conducted by Nicolin et al22.1TIB‐71 and RAW264.7CRL‐2278 were purchased from ATCC. Cell properties are detailed in Table TIB‐71 cells were cultured as our previous studies reported.TIB‐71 was seeded into 96‐well plate by the density of 2 × 103 cells/mL in DMEM, containing L‐glutamine (4 mM), FBS (10%), sodium bicarbonate (1.5 g/L) and glucose (4.5 g/L). Besides that, RAW264.7CRL‐2278 was initially seeded into the 96‐well plate at the same density to RAW264.7TIB‐71 (2 × 103 cells/mL) in RPMI‐1640 (according to ATCC's instruction), which contains L‐glutamine (2 mM), FBS (10%), sodium bicarbonate (1.5 g/L) and glucose (4.5 g/L), HEPES (10 mM) and sodium pyruvate (1 mM) before the osteoclastic induction.RAW264.72.2TIB‐71 as we previously reported.CRL‐2278 in our current study in order to abrogate the culturing divergences with RAW264.7TIB‐71. Specifically, we changed the induction from RPMI‐1640 medium into α‐MEM at the first induction day. Both cell lineages were inducted for 7 days in several culture group with different stimulate conditions, the grouping details are listed in For osteoclastic differentiation, we induced the RAW264.72.3After initially seeding, both cell lineages were allowed to grow and attach the wall for 24 hours. After 24 hours, we starting to treat cells with cocktailed stimulators for 10 days, and we counted this initial treating day as day 0. Subsequently, cellular morphological alterations were recorded and evaluated at days 1, 2, 3, 4, 5, 6 and 7 by an inverted microscope Nikon (Nikon) and NIS‐Element analysis software (Nikon) for image analysis. TRAP staining kits were purchased from Sigma‐Aldrich Co. According to instructions, after PBS washing two cell lineages were fixed by paraformaldehyde (4%) lasting for 30 minutes. Subsequently, both cell lineages were washed by PBS and then stained by TRAP solution at 37°C in 1 hour, the staining results were evaluated by Nikon (Nikon) and NIS‐Element analysis software (Nikon) for image analysis.2.4We performed cytoskeletal fibrous actin (F‐actin) staining for two subjects in order to observe the podosome patterns and sealing zone formation in each cell linages. Briefly, two cell lineages were washed by PBS, after washing cells were incubated in formaldehyde (4%) for 15 minutes fixing, then cells washed by PBS for three times. In order to increase permeability, before the phalloidin red incubation, the Triton X‐100 (0.1%) was added for 1 minutes. After 1 hour of incubation, DAPI was used for nucleus staining. F‐actin–stained cytoskeletons were visualized and quantified with a fluorescence microscope Nikon (Nikon) and NIS‐Element analysis software (Nikon) for image analysis. Besides that, we performed SEM. Briefly, matrices were then fixed by sodium cacodylate (0.1 M) containing glutaraldehyde (2.5% (v/v)); the final cells were recorded by the JSM6700F electron microscope.2.5t test’ methods by the software GraphPad Prism (version 8). Final, the value of P < .05 was defined as statistical significant in our current study, the data presented by the mean ± standard deviation (SD) manner.Final data results were analysed by ‘two‐way ANOVA’ and ‘Student's 33.1CRL‐2278 showed a faster and stronger osteoclast differentiation than RAW264.7TIB‐71 within a 7 days of induction. However, our previous study has showed that RAW264.7TIB‐71 could be induced to gain osteoclasts in a short‐term induction .TIB‐71 could be induced into giant osteoclasts. Moreover, even there were vast studies used the osteoclasts derived from RAW264.7 (RAW‐OCs) for their in vitro osteoclastic studies, there were few reports noted the brand of cell lineages for each usage. Therefore, in our present study we performed a subtle compare for these two cell lineages . However, even there were numbers of fused cells during RAW264.7CRL‐2278 culture, there were no multinucleated cells (nuclei ≥ 3) in the RANKL independently induced group and the LPS + INF‐γ–induced group. However, interestingly, we found that under the fully activated condition (LPS + INF‐γ), the RAW264.7CRL‐2278 could be induced into multinucleated cells by RANKL independently treatment, but the numbers of nuclei are significantly less than RANKL independently induced RAW264.7TIB‐71. These results interestingly demonstrated that, even under the fully activated condition of RAW264.7CRL‐2278, this cell lineage possesses a completely different property comparing to its parental cell lineage RAW264.7TIB‐71. Interestingly, consist with our previous studies, in our present study, we noticed M‐CSF has not shown significantly enhancing effects on osteoclastic formation for both RAW264.7TIB‐71and RAW264.7CRL‐2278  , matured osteoclast adhesion to the bone surface followed cytoskeletal organelles rearrangements. These rearrangements allowed polarization of osteoclasts to form four functional domains, including ruffled border with an isolated milieu of resorptive microenvironment and podosome patterned sealing zone (SZ). In that, SZ is one of the critical functional domains that segregates the resorptive microenvironment between osteoclast adhesion zone and bone tissue. Therefore, we performed F‐actin assay to assess the osteoclastic cytoskeletal SZ structure formation estimating osteoclastogenesis for RAW264.7p Figure ,D.CRL‐2278 in fully activated condition with RANKL‐induced RAW264.7TIB‐71 by SEM. Our results showed the morphological differences between the two cell lineages can be noted, RANKL induced fully activated RAW264.7CRL‐2278 cells shaped in wedge shape with much microvilli in cellular dorsal surface, which in turn the RANKL induced RAW264.7TIB‐71 cells presented an flat and smooth surface in cellular dorsal periphery. Moreover, the SZ could be clearly noted in RANKL‐induced RAW264.7TIB‐71 cells . Therefore, in our current study we conducted an extensive study for these two cells, which provide more details and evidences for RAW‐OC culturing methodology.Osteoclast formation and functional activity are important for bone homeostasis, which is key study target for bone biology.CRL‐2278 failed to form OCs under several stimulating conditions; instead, RAW264.7CRL‐2278 could generate multinucleated cells under the condition of RANKL‐stimulated LPS + INF‐γ group. However, on the contrary to previously reported, comparing the RANKL induced osteoclasts from RAW264.7TIB‐71 osteoclasts from RANKL induced RAW264.7CRL‐2278 presented a significantly small size and less nuclei numbers.Osteoclasts could commonly be characterized by two criteria: the multiple nuclei (≥3) and TRAP activity.CRL‐2278RAW264.7 cell lineage.Image analysis has been a promising tool for quantitation of complex biological processes, and image analysis could enable assessment for the correlation between the osteoclastic activity and the cellular maturation state, which further stratified the treatment efficacies. Therefore, we further performed the immunofluorescence and SEM for investigating the cytoskeleton of these cells. In fact, podosome could be observed in a wide variety of cell types, including macrophages and dendritic cells. An actin‐rich organelle podosome plays critical role in various cellular functions, such as motility, adhesion and matrix invasion. During the osteoclast formation and maturation, podosomes initially are observed from SEM for their localized on ventral membrane of the osteoclast at a contact site against the underlying substrate. During the osteoclastic differentiation, podosomes rapidly pattern into ‘clusters’ and ‘rings’; further with the maturation of osteoclasts, podosomes finally pattern into the SZ. Most importantly, the highly organized SZ of the osteoclasts is a unique apparatus, which could be only observed in matured osteoclasts, and serves for both osteoclastic cellular adhesion and extracellular matrix resorption. Our current study results showed during the whole culturing, there was no SZ patterned in The authors declare that they have no competing interests.Lingbo Kong: Conceptualization ; Data curation ; Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Writing‐original draft . Rui Ma: Investigation ; Methodology ; Writing‐review & editing . Yang Cao: Data curation ; Investigation ; Software . Xiaobin Yang: Methodology . Liang Yan: Conceptualization ; Data curation ; Resources ; Supervision (lead). Yuan Liu: Validation ; Writing‐review & editing . Wanli Smith: Conceptualization ; Methodology .None animal studies have involved in the current study.The manuscript is approved by all authors for publication.Supplementary DataClick here for additional data file.


Acromial anatomy has been found to be correlated with degenerative full-thickness rotator cuff tears in current studies. However, research on the relationship between acromial anatomy and articular-sided partial thickness of rotator cuff tears (PTRCTs) is still lacking. The purpose of this study was to evaluate whether these imaging graphic parameters exhibit any association between acromial anatomy and degenerative articular-sided PTRCTs.Between January 2016 and December 2018, a total of 91 patients without a history of trauma underwent arthroscopy as an articular-sided PTRCT group. In the control group, with age- and sex-matched patients, we selected 91 consecutive outpatient patients who underwent shoulder magnetic resonance imaging (MRI) because of shoulder pain and an MRI diagnosis of only synovial hyperplasia and effusion. MRI was used to measure the acromial type, acromiohumeral distance (AHD), lateral acromial angle (LAA), acromion index (AI), and critical shoulder angle (CSA) by 2 independent observers.P = 0.532, 0.277, and 0.108, respectively). AI and CSA were significantly higher in degenerative articular-sided PTRCTs . A good correlation was found between AI and CSA to measurement(Pearson correlation coefficient = 0.631).The acromion type, AHD and LAA showed no difference between degenerative articular-sided PTRCTs and controls (Our study revealed that higher AI and CSA were found in degenerative articular-sided PTRCTs. Acromial anatomy with a large acromial extension was associated with the occurrence of degenerative articular-sided PTRCTs. Rotator cuff tears (RCTs) are a common shoulder disease and are highly prevalent among elderly people . In addiBigliani et al.  found thThe parameters described above were measured singly or were focused on the full thickness of the RCT. The results are still controversial. Humeral head translation secondary to FTRCT has been reported , while iWe retrospectively evaluated data from consecutive patients at our institution from January 2016 until December 2018 who underwent arthroscopically confirmed isolated articular-sided PTRCTs. The patients we included had grade III articular-sided PTRCTs  curve higher than 0.8 was considered significant. According to previous studies [P value of 0.05 using R statistical environment (v4.4.1) .All statistical analyses were performed using SPSS version 22 software. Interclass correlation coefficients (ICCs) were used to evaluate intraobserver and interobserver consistency in a two-way mixed model, and values greater than 0.75 were considered sufficient for reliability . The χ2  studies , 24–26, All demographic data of the patients are listed in Tables The ICCs of all imaging parameters were reliable, with intraobserver ICCs of 0.911 for AI, 0.979 for CSA, 0.972 for LAA and 0.934 for AHD, with interobserver ICCs of 0.837 for AI, 0.960 for CSA, 0.946 for LAA and 0.875 for AHD.P = 0.003). The mean AI was also significantly higher in articular-sided PTRCTs than in the controls . For CSA in the diagnosis of articular-sided PTRCTs, the area under the ROC curve was 0.624, and for AI in the diagnosis of articular-sided PTRCTs, the area under the ROC curve was 0.646 . The average AHD was also similar between the two groups .The mean LAA values were not significantly different between group 1 and group 2 (78.3 ± 4.97 vs. 79.5 ± 5.43; We found a good correlation between CSA and AI and a fair correlation between LAA and CSA and AI, AHD and CSA, and AHD and AI. Poor correlations were found between AHD and LAA and between age and acromion and the other parameters Table .Table 3The main finding of the study was that statistically significant differences in AI and CSA were found between the articular-sided PTRCT group and the matched control group utilized MRI. Some studies have suggested that FTRCTs originate from partial tears . AlthougAI was first described by Nyffeler et al.  in 2006,In recent years, CSA was first described by Moor et al.  as havinAlthough a hook type seemed to have a higher association with RCTs according to Bigliani et al. , recentlA good correlation between CSA and AI was found in this article, which is similar to a previous study . AccordiSome limitations of this study should be considered. First, potential measurement bias may exist, although two well-trained orthopedists independently made the measurement without a previously known diagnosis. Second, this is a retrospective study in which all patients were symptomatic and underwent surgery, and selection bias may exist owing to asymptomatic articular-sided PTRCTs. Finally, since plain radiographs or computed tomography were not used, the accuracy of MRI measurement was not further verified, which also might have caused a bias.Our study revealed that higher AI and CSA were found in degenerative articular-sided PTRCTs. Acromial anatomy with a large acromial extension was associated with the occurrence of degenerative articular-sided PTRCTs.