The varying period of time before an indolent low-grade glioma (LGG) inexorably transforms into a high-grade glioma (HGG) provides a unique opportunity for intervention. While recent clinical trials have demonstrated a clear survival benefit with early treatment of LGGs rather than watchful waiting, there is still a need to identify prognostic molecular markers that predict malignant transformation ,2. AlthoCX3CR1 has a common polymorphism in which valine at amino acid position 249 is substituted by isoleucine (V249I), conferring a reduced binding affinity to CX3CL1. In a previous study, CX3CR1 V249I polymorphism conferred a significant survival benefit in glioblastoma, in association with reduced numbers of TAMs, although the mechanistic details were incompletely explored [CX3CR1 is a chemokine receptor expressed by TAMs and tumor cells. Its cognate ligand, CX3CL1 or fractalkine, can exist in a membrane-anchored or soluble form, and mediates adhesion or chemotaxis, respectively. In the CNS, CX3CL1-CX3CR1 signaling has been implicated in regulation of microglial recruitment and function in various disease contexts including stroke, Parkinson’s disease, Alzheimer’s disease . CX3CR1 explored .CX3CR1 V249I polymorphism on survival may be more sizeable in LGGs compared to glioblastomas. Indeed, in our cohort of 90 patients with LGGs that ultimately progressed to HGGs, CX3CR1 V249I polymorphism conferred a strong survival benefit, extending median overall survival by 5 years and progression-free survival by nearly 2 years [CX3CR1 V249I polymorphism. Correspondingly, microvessel density was also lower, reflecting reduced angiogenesis.Given the more protracted natural history, we hypothesized that the impact of the  2 years . This efCX3CR1 genotype dependent alterations. CX3CR1 V249I polymorphism was associated with lower transcript levels of CCL2, implicated in TAM recruitment, and MMP9, involved in invasion and angiogenesis. Interestingly, these effects were at least in part mediated through the impact of the CX3CR1 V249I polymorphism on the tumor cells. Several patient-derived glioblastoma stem cell (GSC) lines expressed CX3CR1 at the surface. GSCs derived from patients without the polymorphism responded to CX3CL1 stimulation by upregulating CCL2, whereas this response was not seen in GSCs from patients with the CX3CR1 V249I polymorphism.Bulk tumor RNA sequencing data were examined for Next, these effects of intratumoral CX3CL1-CX3CR1 signaling were recapitulated in a PDGFB-driven mouse model of glioma. Co-expression of human CX3CL1 and CX3CR1 (wild-type) within tumors led to worsened survival and higher histological grade. Further characterization demonstrated increased density of TAMs and microvessel density as well. Taken together, these data suggest that intratumoral CX3CL1-CX3CR1 signaling promotes a proangiogenic and immunosuppressive microenvironment through generation of CCL2 and MMP9, which is abrogated in the presence of CX3CR1 V249I polymorphism.In the CNS, CX3CR1 signaling has been mostly investigated in the context of regulating microglial phenotypes. However, it is also expressed in a wide variety of tumors outside the CNS and promote proliferation, invasion and survival. Our study is the first to characterize the important role for CX3CR1 signaling within the glioma cells to promote an immunosuppressive and pro-angiogenic microenvironment. These results of course do not rule out the likely contribution of microglial CX3CR1 signaling; but intratumoral CX3CR1 signaling was convincingly demonstrated through CX3CL1 mediated CCL2 upregulation in GSCs as well as characterization of phenotypes in CX3CL1/CX3CR1 co-expressing mouse models. Broadly, our data highlight the complex, intertwined nature of the interactions between the tumor and the microenvironment and how gliomas hijack normal signaling pathways. Full understanding of CX3CR1 signaling will require the resolution of single-cell sequencing in human tumors and use of cell-type-specific genetic manipulations in animal models – both of which are currently ongoing in our laboratory.CX3CR1 V249I polymorphism, may play an important role in individualizing therapy, reserving maximal treatment for those LGGs which are likely to behave more aggressively.A recent clinical trial demonstrated the clear survival advantage of adjuvant chemotherapy and radiation compared to radiation alone following surgical resection of LGGs . However


Oral cancer represents the sixth most common cancer in the world and is associated with 40-50% survival at 5 years. Within oral malignancies, oral squamous cell carcinoma (OSCC) is commonly preceded by potentially malignant lesions, which, according to histopathological criteria, are referred to as oral dysplasia and their diagnosis are associated with higher rates of malignant transformation towards cancer. We recently reported that aberrant activation of the Wnt/β‑catenin pathway is due to overexpression of Wnt ligands in oral dysplasia. However, the expression of other regulators of this pathway, namely components of the β-catenin destruction complex has not been explored in oral dysplasia. Using immunohistochemical analyses, we evaluated nuclear expression of β‑catenin and its association with Wnt3a and Wnt5a. Likewise, components of the β-catenin destruction complex, including Adenomatous Polyposis Coli (APC), Axin and Glycogen Synthase Kinase 3 beta (GSK-3β) were also evaluated in oral dysplasia and OSCC biopsies. We found that moderate and severe dysplasia samples, which harbored increased expression of nuclear β‑catenin, depicted augmented cytoplasmic expression of GSK‑3β, Axin and APC, in comparison with OSCC samples. Also, GSK-3β was found nuclear in mild dysplasia and OSCC samples, when compared with other study samples. Cytoplasmic levels of components of the β-catenin destruction complex are increased in oral dysplasia and might be responsible of augmented nuclear β‑catenin. Key words:Oral cancer, oral dysplasia, β-Catenin, Wnt ligands, destruction complex. Oral carcinogenesis is a multifactorial process that involves several molecular and genetic alterations . Oral sqOSCC is commonly preceded by potentially malignant lesions in the oral mucosa, which are clinically known as leukoplakia and erythroplakia, and are histologically classified as oral dysplasia . The diaThe Wnt/β-catenin signaling pathway plays an important role in cell growth, differentiation, migration and proliferation . β-catenin vitro cell cultures, respectively  for antigenic recovery and then washed with PBS for 5 min. Endogenous peroxidase activity was blocked by incubating the sections with 3% hydrogen peroxide at room temperature for 10 min. Sections were pre-incubated with horse serum for 30 min at room temperature and incubated with primary antibodies overnight at 4°C. Monoclonal anti-β-catenin  was from DAKO , monoclonal antibodies raised against APC , Axin , Wnt5a  were from Santa Cruz Biotechnology . Anti-Wnt3a  was from Abcam , while mouse monoclonal anti‑GSK-3β  was from BD Transduction Laboratories . After washing the sections with PBS for 5 min, they were incubated with biotinylated secondary antibodies for 30 min at 37 °C and with peroxidase-conjugated streptavidin  for 30 min at 37°C. The reaction was visualized with diaminobenzidine (DAB) and stained with Harris Hematoxylin. Negative controls were obtained by using PBS instead of primary antibodies.Positive controls were used to determine the ideal concentration for each antibody used in this study. For β-catenin, a colon tissue sample was used; for Wnt3a, a kidney tissue sample was used as positive control; For Wnt5a, a melanoma sample; for APC, a colon tissue sample; for Axin, using a skin sample as positive control; finally, for GSK-3β, an ovary sample was used as positive control.Sample Analysis. Under microscopic field of 400-fold magnification, the cellular localization of antigens was classified as membranous, cytoplasmic or nuclear, depending on the immunolocalization pattern. Tissues were classified as nuclear positive for β-catenin, if more than 10% of cells showed nuclear staining. Quantification of nuclear immunostaining was performed by analyzing five randomly selected fields per sample, reaching a total of 1000 epithelial cells per sample. The number of cells with positive staining was determined by using the Image J program. Intensity of staining was assessed as mild (+) when the positive staining was achieved within the range of 1-25% of the cells analyzed, while staining was considered as moderate (++) or intense (+++) when positive staining was obtained within the range of 25-75% and 75-100% of the cells analyzed, respectively. In non-homogeneous cases, the predominant intensity (over 75% of cells) was recorded. All immunohistochemical analyzes performed in this study were carried out by two expert observers in the area. The fields were photographed using an Olympus CX41 microscope using the software Micrometrics SE Premium program.- Statistical analysisp <0.05) was accepted as statistically significant. All statistical tests were performed using Stata 11.0 software.Shapiro-Wilk test was used for an exploratory normality analysis of the data. ANOVA with a Bonferroni post-hoc test was used for statistical comparison between samples. A value of significance of 5% or less . Similarly, severe oral dysplasia samples showed predominant nuclear localization of β‑catenin when compared with OSCC samples, where a cytoplasmic location of the antigen was frequently observed (p < 0.01)  Fig. . These r01) Fig. ,10. The - Increased expression of Wnt3a in samples of severe oral dysplasiap <0.01)  , 100% of epithelial cells expressed Wnt3a in all epithelium strata but, unexpectedly, it was not expressed in the healthy oral mucosa (n=3) Fig. . Interes01) Fig. . As wellThe expression of Wnt5a also was analyzed, however no differences were found between the study groups (data not shown). This suggests that that Wnt3a, but not Wnt5a, is required for Wnt signaling activation at advanced dysplasia stages. These results support previous observations where secretion of Wnt3a is required for the activation of the Wnt/β‑catenin and the expression of genes in oral dysplasia .- Increased expression of APC and Axin in severe oral dysplasia samples In the healthy oral mucosa samples, APC was located at the cytoplasmic level Fig. . Howeverp <0.05).No differences were found in APC intensity in other study groups. Expression and localization of APC are summarized in Fig. Also, moderate and severe dysplasia samples showed higher intensity of APC than mild dysplasia and healthy oral mucosa samples (p <0.05)  Fig. . Express- Increased levels of nuclear GSK-3β in OSCC samples compared to oral dysplasia samplesp <0.05).All oral dysplasia samples showed cytoplasmic GSK-3β localization. Interestingly, two mild dysplasia samples showed both cytoplasmic and nuclear localization of GSK-3β (12% of nuclear positivity), as well as absence of nuclear expression of β-catenin (Fig. Like in oral dysplasia samples, OSCC samples also displayed a cytoplasmic localization of GSK-3β. Curiously, 4 well-differentiated OSCC samples and 6 moderate/poorly differentiated OSCC samples showed both cytoplasmic and nuclear expression of GSK-3β, as well as absence of nuclear expression of β-catenin. The samples of well-differentiated OSCC showed a 20% of nuclear positivity, while in moderate/poorly differentiated OSCC a 40% of nuclear positivity for GSK-3β was observed (Fig. in vitro and ex vivo models (in vitro approach to propose a mechanism where the endosomal sequestration of the β-catenin destruction complex may be key for nuclear β-catenin translocation in oral dysplastic cells (Aberrant activation of Wnt/β-catenin is commonly associated with malignant progression in several cancers . We haveo models . Howeveric cells . DespiteIt is known that increased expression of nuclear β-catenin is associated with moderate and severe oral dysplasia, while decreased membrane expression of β-catenin is associated with worse prognosis of OSCC ,18,19. IAberrant activation of the β-catenin pathway has been associated to increased secretion of Wnt ligands, leading to augmented translocation of β‑catenin to the nucleus in different types of cancer ,20-22. Aet al., reported in colon cancer cell lines that nuclear GSK-3β decreased nuclear β‑catenin activity (It is known that the activation of Wnt signaling through ligand-receptor binding inhibits GSK-3β‑mediated phosphorylation of β-catenin, leading to cytoplasmic accumulation of β-catenin and translocation to the nucleus . Some stactivity . Thus, oIn summary, we propose that nuclear detection of β-catenin in oral dysplasia is associated to the activation of Wnt signaling pathway by the Wnt3a ligand. This leads to nuclear accumulation of β-catenin and cytoplasmic increase of the components of the β-catenin destruction complex in dysplasia. However in OSCC, nuclear GSK-3β may behave as a negative regulator of nuclear β-catenin, promoting tumorigenesis by Wnt-independent routes.


CTNNB1); and (2) promoter mutations in telomerase reverse transcriptase (TERT). Here, we review recent findings regarding the interplay between TERT and β-catenin in order to better understand their role in liver cancer.Liver cancer is one of the deadliest human cancers. Two of the most common molecular aberrations in liver cancer are: (1) activating mutations in the gene encoding β-catenin (TERT) promoter (TERTp) and CTNNB1 gene encoding β-catenin are widespread in HCC . TERTp mutations are predicted to increase TERT transcription and telomerase activity. This review focuses on exploring the role of TERT and β-catenin in HCC and the current findings regarding their interplay. TERT can have contradictory effects on tumorigenesis via both its canonical and non-canonical functions. As a critical regulator of proliferation and differentiation in progenitor and stem cells, activated β-catenin drives HCC; however, inhibiting endogenous β-catenin can also have pro-tumor effects. Clinical studies revealed a significant concordance between TERTp and CTNNB1 mutations in HCC. In stem cells, TERT acts as a co-factor in β-catenin transcriptional complexes driving the expression of WNT/β-catenin target genes, and β-catenin can bind to the TERTp to drive its transcription. A few studies have examined potential interactions between TERT and β-catenin in HCC in vivo, and their results suggest that the coexpression of these two genes promotes hepatocarcinogenesis. Further studies are required with vertebrate models to better understand how TERT and β-catenin influence hepatocarcinogenesis.Hepatocellular carcinoma (HCC) is one of the deadliest human cancers. Activating mutations in the telomerase reverse transcriptase ( TERT), TP53 (tumor protein p53), and CTNNB1 encoding β-catenin are the top three most frequently mutated genes in HCC, altered in 47.1%, 29.2%, and 27.4% of cases from large-scale sequencing projects . TER. TERTERTpression  [5]. Thend Spain .CTNNB1 mutations appear to be the initiating event, being found in 20% of HCA without malignant transformation (0% with TERT mutations)   105]. β. βCTNNB1subtypes ,107. In utations  .ormation ,109,110.TERTp and CTNNB1 mutations, as 57.6% of CTNNB1-mutated HCC had TERTp mutations  [TERTp mutations and WNT pathway alterations  in HCV and non-virus related HCCs  [A study done on a European cohort with a high prevalence of HCV-related HCC also showed the coexistence of  0.4192) . AnotherTERTp mutations are very high and more frequent than in HCC associated with HBV or even HCV [CTNNB1 mutations were mutated for TERTp; however, the association was statistically insignificant , likely due to the small sample size of 11 [In HCC cases arising in patients with non-alcoholic fatty liver disease (NAFLD), even HCV ,54. In aTERTp and β-catenin mutations; however, none of them found evidence to support the direct regulation of one gene by the other [TERTp mutations and not CTNNB1 mutations, β-catenin target genes (GLUL and LGR5) were not upregulated compared to HCC without mutations in TERT or β-catenin [CTNNB1 mutations, TERT transcription was not increased [The aforementioned studies all demonstrated a high concordance between he other ,54,103. TERTp and β-catenin mutations. Studies done on Taiwanese, Korean, and Chinese HCC cohorts did not reveal any concurrence between the two mutations [TERTp mutations in NAFLD-HCC patients [TERTp and CTNNB1 mutations tended to have a larger sample size than studies that did not identify a significant correlation  led to a malignant phenotype of loss of contact inhibition, increased anchorage-independent growth, and improved ability to form tumors in athymic nude mice [TERT and TERC, this study showed that telomerase activity is not required for the short-term expansion of transformed cells [The strong correlation between ude mice . These Fude mice . This fied cells .TERT and β-catenin in HCC with an elegant in vivo screen [Tert and CTNNB1 developed HCC, compared to zero of six mice overexpressing CTNNB1 alone [n = 9), no Tert; CTNNB1 mice developed liver tumors [Tert and β-catenin may be insufficient for HCC, and additional spontaneous genetic alterations may be required [TERT and CTNNB1, will be critical for confirming and extending the results of Molina-Sánchez et al. Catalytically inactive TERT (TERTci) constructs can be particularly useful in determining whether interactions between TERT and β-catenin require the telomerase activity of TERT or act through non-canonical mechanisms.Molina-Sánchez et al. showed cooperation between o screen . They usB1 alone . Howeverr tumors . The autrequired . Future TERTp and CTNNB1 mutations are concordant in patient HCC; (2) in animal models, overexpression of TERT or CTNNB1 promotes HCC, although the effect is modest in most models and often requires another oncogene or insult; (3) in cultured stem cells, TERT and CTNNB1 cooperate via direct or indirect interactions during the transcription of target genes; and (4) both CTNNB1 and, to a lesser extent, TERT, have key roles in cellular homeostasis, making the potent inhibition of endogenous genes unrealistic from a therapeutic perspective. The key points from the above studies are: (1) TERTp and CTNNB1-mutated HCC, including: (1) targeting non-canonical effects of TERT, critical for its tumor-promoting interactions with β-catenin; (2) targeting the interaction between TERT and β-catenin; and (3) combining relatively low doses of Wnt/β-catenin pathway and TERT inhibitors.Using cultured human liver cancer cells and vertebrate HCC models will be critical for confirming that the mechanism of TERT/β-catenin interaction identified in stem cells applies to HCC and establishing that non-canonical effects of TERT drive its interactions with β-catenin and the resulting HCC-promoting effects. Delineating exactly how TERT and β-catenin interplay would suggest promising treatment strategies for CTNNB1.With respect to the latter approach, several drugs targeting either the WNT/β-catenin pathway or TERT are in early clinical trials or preclinical testing. The porcupine inhibitor CGX1321 and the Frizzled8 decoy receptor OMP-54F28 are currently in Phase I clinical trials ,115. TheOne option for inhibiting WNT/β-catenin signaling further downstream is to block β-catenin’s interactions with its transcriptional partners to prevent the expression of target genes. To that effect, molecules targeting β-catenin binding to CREB-binding protein (CBP) (PRI-724 (Phase 1 and 2)) or to TCF/LEF elements  are being tested ,115. CBPCurrent strategies for the therapeutic targeting of TERT include: (1) small molecule inhibitors against TERT, like BIBR1532 ; (2) genCTNNB1 and TERTp mutations in HCC presents attractive diagnostic and prognostic possibilities. Risk stratification could be done based on the clues we have regarding the temporal play of gatekeeper and driver oncogenes. TERTp mutations are the first changes associated with neoplasia in cirrhotic livers. Thus, they could potentially be used to predict HCC risk in cirrhotic patients, just as β-catenin activation is currently used to predict HCC risk in patients with HCA. Further studies examining the prevalence of TERTp mutations in cirrhotic patients without HCC would be required to determine the feasibility of this approach.The association of TERTp and CTNNB1 mutations, with response to specific treatments to guide future patient treatment regimens.HCC poses a therapeutic challenge due to the late diagnosis and paucity of effective treatments for advanced disease. The molecular heterogeneity in HCC further exacerbates this challenge, highlighting the need for increased identification and understanding of biomarkers and their prognostic and therapeutic value. Current HCC treatments are not tailored to patients based on the molecular features of their tumors, as no genetic perturbations have been definitively shown to influence therapeutic response in patients. As the armamentarium of approved HCC therapeutics expands, it will be critical to correlate molecular subtypes, including those with


The protein arginine methyltransferases (PRMTs) are involved in such biological processes as transcription regulation, DNA repair, RNA splicing, and signal transduction, etc. In this study, we mainly focused on PRMT5, a member of the type II PRMTs, which functions mainly alongside other interacting proteins. PRMT5 has been shown to be overexpressed in a wide variety of cancers and other diseases, and is involved in the regulation of Epstein-Barr virus infection, viral carcinogenesis, spliceosome, hepatitis B, cell cycles, and various signaling pathways. We analyzed the regulatory roles of PRMT5 and interacting proteins in various biological processes above-mentioned, to elucidate for the first time the interaction between PRMT5 and its interacting proteins. This systemic analysis will enrich the biological theory and contribute to the development of novel therapies. Protein post-translational modification (PTM) is critical for proteome diversification, which contributes to the regulation of protein function and cell signal transduction. Among PTM, the protein methylation, modified by protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs) targeting at lysine and arginine residues, respectively, has attracted attention, , howeverIn eukaryotes, monomethylation, and dimethylation modification of arginine, are correspondent to the formation of monomethylated arginine, symmetrical dimethylarginine and asymmetric dimethylarginine . At presMethylation either changes the structure of chromatin and leads to chromatin remodeling, or provides binding sites for transcriptional regulators . The metAmong the nine members of the PRMT family, PRMT5, PRMT1, and CARM1 are most highly expressed in cancer . PRMT5 i1, IntAct2, MINT3, HPRD4, PINA5, and BioGRID6. STRING covers the largest number of organisms and uses the widest breadth of input sources, including automated text-mining and computational predictions  is characterized by specifically infecting humans and some primate B cells ormation . In addiormation .Studies also found that EBV utilizes PRMT5 to target and silence genes with important tumor suppressor properties during B-cell immortalization. PRMT5 overexpression may be an important mechanism to promote the immortalization of B cells after EBV infection . In addiInflammation might contribute to the development and progression of malignancies . PRMT5 pPRMT5 is overexpressed in hepatocellular carcinoma (HCC), and associated with aggressive clinicopathological parameters, such as poor differentiation, higher incidence of hepatic vein invasion, larger tumor size, higher AFP levels and worse prognosis . PRMT5 iIn humans, more than 95% of multi-exon precursor mRNA transcripts undergo alternative splicing. As a splicing modulator, PRMT5 mediates SDMA of the spliceosome Sm protein . The Sm Study shows that pICln recruits Sm proteins into the PRMT5 complex for methylation, then the methylated Sm proteins are loaded onto snRNAs to form snRNPs . snRNP aIn the human breast cancer MDA-MB-231 cell, PRMT5 and WDR77 show high expression and nuclear localization. ZNF326 is a zinc finger protein that moves with the core mRNP particles during transcription and regulates splicing by controlling the elongation rate of transcription. ZNF326 interacts with PRMT5 and WDR77; HNRNPH1, a component of mRNP particles, also interacts with PRMT5/WDR77 complexes. The loss of PRMT5 and WDR77 leads to the overall defect of alternative splicing, which is mainly due to the change of exon inclusion or exclusion through exon skipping (SE),  and thisPRMT5 regulates cell cycle and apoptosis by the following aspects.Firstly, PRMT5 regulates cell cycle and apoptosis by methylating histones. In acute lymphoblastic leukemia (ALL), PRMT5 promotes the proliferation of lymphocytes by inhibiting apoptosis, and the down-regulation of H4R3sme2 by PRMT5 silencing induced ALL differentiation from the pre-B to immature B stage . In addiSecondly, PRMT5 functions in a DNA damage responsive co-activator complex that interacts with p53, and is responsible for the methylation of p53. PRMT5 depletion caused an increase of sub-G1 cells during the DNA damage response, simultaneously, compared with wild-type p53, a p53 mutant compromised in arginine methylation may affect the cell cycle . In addiThirdly, PRMT5 promotes cell cycle progression by up-regulating cell cycle proteins such as β-catenin, Cyclin D1, CDK4/6, and CCND1/D2/E1 . PRMT5 iL which is a key anti-apoptotic protein. PRMT5 and PRMT1 regulate the polyubiquitination and degradation of CFLARL by influencing the interaction between CFLARL and ITCH, then regulate the apoptosis of non-small cell lung cancer cells by regulating CFLARL , regulates the transcription of LCE1C gene, PRMT5 interacts with LCE1C, and LCE1C promotes the cytoplasmic retention of PRMT5 or the transport of PRMT5 from nucleus to cytoplasm through direct interaction, thereby preventing the nuclear function of PRMT5 . It is wSecondly, PRMT5 is involved in immune hematopoietic regulation. PRMT5 is essential for B cell development. It regulates the fidelity of B cell transcription and splicing, protects B cell from apoptosis and promotes proliferation . The depThirdly, PRMT5 is involved in tumor angiogenesis, cell invasion and metastasis. PRMT5 interacts directly with HOXC10, promoting the enrichment of H3R2me1 and H3R2me2s on the VEGFA promoter, and then recruiting WDR5 for subsequent methylation of H3K4 on the VEGFA promoter, which eventually leads to the transcription of VEGFA and promotes angiogenesis in glioma . PRMT5 pFourthly, PRMT5 is involved in the regulation of homeostasis. Cells subjected to genotoxic stress need to reestablish reduction-oxidation (redox) homeostasis to scavenge genotoxic stress-induced reactive oxygen species (ROS). PRMT5 contributes epigenetically to JDP2/β-catenin-activated GSH metabolism upon genotoxic stress . In addiTo sum up, the summarized model diagram of PRMT5 functions is shown in 7, GSK3326595, a substrate competitive inhibitor, currently in the stage of phase II clinical research and JNJ64619178, a SAM (S-adenosyl-l-methionine) mimetic/competitive inhibitor, in phase I clinical stage, and Pfizer’s target drug PF-06939999, also in phase I clinical stage. The rational design of targeted therapy combined with PRMT5 inhibitors has become an effective treatment strategy. Some penitential strategies have been reported as follows.Due to the key function of PRMT5 and PIPs in various biological processes, PRMT5 is supposed to act as a therapeutic target. Currently, there is no PRMT5 inhibitor approved for marketing anywhere in the world. Three molecules have entered clinical trials for multiple cancer typesThe enzymatic activity of PRMT5 is necessary for the regulation of PPARα and PGC-1α expression and mitochondrial biogenesis. Inhibition of PRMT5 is beneficial to β-oxidation and helps reverse liver steatosis. Targeting PRMT5 may have therapeutic potential for the treatment of fatty liver . MeanwhiPRMT5 inhibitors can inhibit the proliferation, activity and function of CD8 + T cells by up- regulating p53 and weakening Akt pathway, moreover, these inhibitors not only damage tumor cells, but also resist tumor immune response . InhibitAt present, inhibition of PRMT5 has also become a potential therapy for methionine adenosine phosphorylase (MTAP)-deficient cancers. In MTAP deficient malignant mesothelioma cells, PRMT5 knockdown reduces the expression of E2F1 involved in cell cycle process, and reduces the factors related to epithelial mesenchymal transition. Targeting PRMT5 may represent a promising new treatment strategy for MTAP deleted malignant mesotheliom .Breast cancer progression, treatment resistance and relapse are thought to originate from breast cancer stem cells (BCSC). PRMT5 is identified as the key modulators for BCSC proliferation and self-renewal. PRMT5 depletion or PRMT5 inhibition will significantly reduce the number of BCSC. The use of small molecule inhibitors of PRMT5 or the downstream targets may be an effective strategy to eliminate this carcinogenic population .In castration-resistant prostate cancer (CRPC), PRMT5 recruits pICln to the androgen receptor (AR) promoter to activate AR transcription, and PRMT5 may be a novel target for CRPC treatment by suppressing expression of AR .Arginine methyltransferase inhibitor 1 (AMI-1), a small molecule inhibitor of PRMTs, could enhance the therapeutic effect of cisplatin in lung cancer cells and exhibited protective ability on normal lung epithelial cells. Therefore, the use of AMI-1 to inhibit PRMT5 is a promising therapeutic candidate for improving lung cancer resistance to cisplatin .Since non-tumor cells express low levels of PRMT5, specific inhibitors of PRMT5 activity that have been developed may show limited cytotoxicity and high specificity against cancer cells, which may facilitate their use of single drug or combination therapy in new cancer treatment methods that are not responsive to conventional therapy .Inhibition of PRMT5 significantly reduces the renal injury and promotes the proliferation of renal tubular cells, at the same time, it reduces the oxidative stress induced by renal ischemia/reperfusion, activates Nrf2/HO-1 signal transduction, and reduces ROS-induced inflammation and apoptosis to prevent ischemia/reperfusion injury . PRMT5 iAt present, our knowledge about the mechanism of PRMT5 dysregulation in diseases, especially cancer, is still limited. This study elucidates for the first time the overall functions of PRMT5 together with interacting proteins; the systemic analysis will enrich the biological theory and benefit the development novel PRMT5-targeting therapies.The original contributions presented in the study are included in the article/ZL was in charge of the writing of manuscript. CW and HJ were in charge of editing the manuscript. SM was in charge of the revision of manuscript. XL conceived and designed this study, reviewed the manuscript and was responsible for the overall content as guarantor. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that has no effective treatment. Nucleolin targeting by the pseudopeptide N6L inhibits the tumor growth and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here, we explored the pathways regulated by nucleolin and N6L in PDAC. We demonstrated that both interact with β-catenin, and that the Wnt/β-catenin pathway is activated in PDAC mouse models. Nucleolin inhibition decreases Wnt/β-catenin pathway activation in PDAC cells and tumors, and represents a new druggable pathway regulated by nucleolin.Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and resistant cancer with no available effective therapy. We have previously demonstrated that nucleolin targeting by N6L impairs tumor growth and normalizes tumor vessels in PDAC mouse models. Here, we investigated new pathways that are regulated by nucleolin in PDAC. We found that N6L and nucleolin interact with β-catenin. We found that the Wnt/β-catenin pathway is activated in PDAC and is necessary for tumor-derived 3D growth. N6L and nucleolin loss of function induced by siRNA inhibited Wnt pathway activation by preventing β-catenin stabilization in PDAC cells. N6L also inhibited the growth and the activation of the Wnt/β-catenin pathway in vivo in mice and in 3D cultures derived from MIA PaCa2 tumors. On the other hand, nucleolin overexpression increased β-catenin stabilization. In conclusion, in this study, we identified β-catenin as a new nucleolin interactor and suggest that the Wnt/β-catenin pathway could be a new target of the nucleolin antagonist N6L in PDAC. Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal cancers. The incidence rate continues to increase for this type of cancer, and only 9% of PDAC patients survive beyond 5 years , often dNucleolin is overexpressed in several tumors and in pancreatic ductal adenocarcinoma (PDAC); 74.5% of patients have a high level of this protein’s expression, and it correlates with a poor prognosis ,3. NucleThe Wnt/β-catenin signaling pathway has a central role in embryogenesis and is dysregulated in several types of cancers ,15. It eWe are investigating molecular pathways in order to find new diagnostic markers and therapeutic targets in PDAC, in order to develop more effective therapies. N6L localizes to tumor tissue and reduces tumor growth by inducing apoptosis and inhibiting cell proliferation ,5. HowevThe nucleolin antagonist N6L inhibits PDAC tumor growth by impairing tumor cell proliferation and survival . We sougp-value  and a control gene (Renilla luciferase). Wnt3A was prepared from the conditioned media of L-Wnt-3A cells, produced, and recovered as described by Shibamoto et al. [β-catenin is important for carcinogenesis and tumor progression of PDAC  . A N6L no et al. . All celo et al. ,32,33. To et al. ,32,33 , enriched by many growth factors and Wnt3A-CM, as previously described in published protocols ,38. TumoOrthotopic pancreatic tumors were induced by injecting mPDAC cells in FVB/n mice, and were treated with N6L 7 mg/kg, as previously established and described in methods . Mice weNucleolin overexpression in human PDAC tumors is a negative prognostic factor for patients, and nucleolin inhibition by N6L inhibits PDAC growth and metastasis in tumor-bearing mice . This woThe study of the pull-down of N6L in PDAC cells allowed us to identify 45 proteins by mass-spectrometry. Nucleolin was present in the N6L pull-down, validating this approach for studying interactors of N6L. A total of 40% of the proteins of the N6L pull-down are known interactors of nucleolin, and 16% belong to the biological processes in which nucleolin is involved (regulation of rRNA processing and positive regulation of mRNA splicing via the spliceosome). We validated β-catenin, the key protein of the Wnt/β-catenin pathway, as a new interactor of N6L and nucleolin by Western blotting in different PDAC cell lines. We also demonstrated an interaction of nucleolin and β-catenin in different human PDAC cells through immunoprecipitation studies. PDAC cells were stimulated with Wnt3A, the isoform commonly used to activate the Wnt/β-catenin pathway ,34. The The importance of the Wnt/β-catenin pathway is well known in tumor progression, invasion, and chemoresistance . PDAC orWnt inhibitors inhibit the Wnt/β-catenin pathway and cancer cell growth through the impairment of β-catenin stabilization ,53,54. SIn this work, we showed that N6L inhibits the Wnt/β-catenin signaling pathway in PDAC cell lines, tumor-derived 3D culture, and mouse models by preventing the stabilization of β-catenin. Nucleolin could promote the stabilization of β-catenin by interfering with the activity of GSK3β, which normally leads to β-catenin degradation. N6L, by inhibiting nucleolin, it may promote the degradation of β-catenin, leading to the inhibition of the Wnt/β-catenin pathway.Murine pancreatic cancer cells (mPDAC) were cultured as previously described  , and 50%3 MIA PaCa-2 cells/well were plated in 96-multiwell plates. The day after, the cells were treated with 10 µM N6L, 30 µM N6L, 1 µM Gemcitabine (Mylan SAS France), or 20 µM IWR-1-endo  in the presence or absence of Wnt3a-CM 1:2 for 24 and 72 h. Cell viability was analyzed by Alamar Blue Assay, as previously described [5 MIA PaCa-2 cells/well plated in 6-multiwell plates. Cell apoptosis was analyzed by incorporating Annexin V  with 1.5 × 105 MIA PaCa-2 cells/well plated in 6-multiwell plates. To determine possible additive and synergistic effects when using combinations, the data from cell viability assays were analyzed using the freely available software, Combenefit (High Single Agent model) [A total of 5 × 10t model) . The sof6 MIA PaCa-2 cells were plated in 100 mm Petri dishes and treated for 24 h with Wnt3a-CM 1:2, with or without 30 µM N6L. Cells were lysed and total RNA was purified using a PureLink RNA Mini Kit , according to the manufacturer’s instructions. Total RNA (100 ng) was reverse-transcribed using hexamer random primers and a first-strand cDNA synthesis kit , and the synthesized cDNA was analyzed by qPCR using FastStart Universal SYBR Green Master . Primers used: AXIN2  and β-ACTIN .1 × 104 MIA PaCa-2 cells/well were plated in 6-multiwell plates. The day after, cells were transfected for 72 h with 20 nM NCL siRNA  for nucleolin or luciferase control siRNA , by using Lipofectamine RNAiMAX . For plasmid transfection, 1.2 × 106 MIA PaCa-2 cells/well were plated in 6-multiwell plates and transfected for 72 h with 5 µg NCL-GFP (nucleolin) or empty-GFP (control), provided by E.M. Reyes-Reyes , using Lipofectamine 3000 (Thermo Fisher).For siRNA transfection, 8 × 105 mPDAC, MIA PaCa-2, and Panc-1 cells/well were seeded in 6-multiwell plates. A Cignal TCF/LEF Reporter Assay Kit , containing Firefly and Renilla plasmids, was used according to the manufacturer’s instructions, using Lipofectamine 3000 (Thermo Fisher). The activities of both Firefly and Renilla luciferase reporters were determined 72 h after transfection and 24 h after treatment with Wnt3a-CM and 30 µM N6L alone or together, using a Dual-Luciferase Assay Kit  according to the manufacturer’s instructions. The reporter activity is presented as the relative ratio of Firefly luciferase activity to Renilla luciferase activity.A total of 8 × 106 MIA PaCa-2 and Panc-1 cells were plated in 75 cm2 flasks. The day after, cells were stimulated or not with Wnt3a-CM for 3 h, and lysed using the same lysis buffer as for the pull-down. One mg of proteins of the extra-nuclear fraction was incubated with 5 µg rabbit anti-nucleolin antibody (Abcam ab22758) or 5 µg control rabbit IgG  for 1 h at 4 °C by mixing samples. Protein G sepharose beads  were prepared and added to the protein/antibody solution, according to the manufacturer’s instructions. After washing, the proteins were eluted and analyzed by Western blotting.A total of 8 × 105 MIA PaCa-2 cells were plated in 6-multiwell plates. The day after, cells were treated for 3 or 24 h with 1:2 Wnt3a-CM, with or without 30 µM N6L. Otherwise, cells were treated for 30 min with 10 µM TWS-119 (Santa Cruz), with or without 30 µM N6L. Cells were lysed with Laemmli buffer, and protein samples were separated by SDS-PAGE and transferred onto a 0.45 μM Immobilon-P membrane (Millipore) following standard protocols. Membranes were blocked with 5% w/v BSA  in TBS 0.1% TWEEN-20  and incubated with the indicated antibodies. Immunocomplexes were visualized by using the luminescence blotting substrate ECL Pierce (Roche). Chemiluminescence was imaged by an imager system . Signal quantification was undertaken with Image Studio Lite (version 5.2), and expressed as the fold change relative to control. The original western blotting figures can be found in A total of 4 × 103 cells/mouse in 50 µL PBS) were injected orthotopically into the pancreas of FVB/n syngeneic mice, as previously described [3 [mPDAC cells (1 × 10escribed . One weeribed [3 . The micribed [3 .7 cells/mouse in 50 µL) were injected orthotopically into the pancreas of immunodeficient mice. The mice were sacrificed after 4 weeks and tumor-derived 3D culture were established by following Tuveson’s laboratory protocol [w/v) collagenase XI (Sigma) and 0.012% (w/v) dispase  in DMEM media containing 1% FBS (Gibco), and the resulting cell suspension was incorporated into growth factor-reduced Matrigel  to obtain a 3D culture.MIA PaCa-2 cells  in 10% growth factor-reduced Matrigel (Corning). After 48 h, Tumour-derived 3D cultures were treated or not with 30 µM N6L for 24 h, were fixed in 4% paraformaldehyde (Thermo Scientific), permeabilized with 0.1% Triton-x100 (VWR), and blocked with 10% donkey serum (Jackson). Primary antibodies and secondary antibodies  were diluted in in 0.3% BSA and incubated at 37 °C in a humid chamber for 1 h. Immunofluorescence pictures were acquired using a DSU IX81 spinning-disc confocal inverted microscope  equipped with a 40× oil objective. Analysis and quantification of active β-catenin and cyclin D1 mean intensity was undertaken with ImageJ software.Immunofluorescence of 5 µM mouse tumor slices from paraffin-embedded blocks was performed as previously described  by usingImmunohistochemistry was performed on 5 µM mPDAC sections, dewaxed, and stained for Ki67 , cleaved caspase-3 , and β-catenin . Antigen retrieval was performed with Dewax and HIER Buffer L (Thermo Scientific TA-999-DHBL) for 20 min at 95 °C. Slices were blocked with BLOXALL Endogenous Blocking Solution  for 10 min, and saturated with Blocking Solution  for 5 min at room temperature. Sections were incubated with primary antibodies diluted in Antibody Diluent (Zytomed ZUC025) for 1 h, and with an ImmPRESS HRP Horse Anti-Rabbit IgG Polymer Detection Kit (VECTOR Laboratories MP-7401) for 30 min, and with ImmPACT DAB (VECTOR Laboratories SK-4105) for 10 min at room temperature. Images were acquired with a NanoZoomer S360 digital slide scanner .n ≥ 3). For continuous variables, we first tested both normality and equality of variance. In the case of a normal distribution, parametric statistical tests were used; otherwise, we used non-parametric tests. For two-group comparisons, we analyzed the data using the two-tailed Student t-test. For multiple group comparisons, 1-way ANOVA Rank with the Dunn method was used .Statistical analyses were performed by using GraphPad Prism  (version 6). The bars represent the mean ± SEM . We claim here, for the first time, that both N6L and nucleolin interact with β-catenin, and that nucleolin targeting by N6L or siRNA leads to the inhibition of the Wnt/β-catenin pathway by preventing β-catenin stabilization in human PDAC cell lines. These results have been validated in tumor-derived 3D culture and a PDAC mouse model.


OCT-4 in β‐catenin-silenced SCC-55 cells. Altogether, Wnt/β‐catenin signaling could potentially target anti-cancer drugs to induce apoptosis and achieve a better clinical outcome.Wnt/β‐catenin signaling is an evolutionarily conserved pathway and plays a crucial role in regulating cancer cell proliferation and tumorigenesis. However, the molecular mechanism behind the Wnt/β‐catenin signaling-mediated carcinogenesis and apoptosis resistance in oral squamous cell carcinoma is not well characterized so far. In the present study, we have investigated the effect of β‐catenin depletion of the perversely activated Wnt/β-catenin signaling pathway on apoptosis resistance and tumorigenesis of the human OSCC cell line SCC-55. RT-PCR and western blot analysis demonstrated that the Wnt/β-catenin signaling pathway and its downstream targets such as DKK1 and AXIN2 are aberrantly activated in SCC-55 cells. Furthermore, upon silencing (RNA interference) of β‐catenin in SCC-55, cells became more sensitive toward the chemotherapeutic drugs and thus resulted in apoptotic cell death. Meanwhile, flow cytometry analysis confirmed the enhanced apoptosis and activation of caspases in β‐catenin RNAi cells. Besides ensuing β-catenin–siRNA transfection, the cell proliferation and cancer colony generating efficiencies are significantly impeded compared to the non-transfected cells. Furthermore, the tumorigenicity was inhibited by the downregulation of  OSCC The irrevocable genetic and epigenetic modifications in oral cancers lead to distorted protein expression and functions, such as Notch1, EGFR, cyclin, p53, pRB, and Wnt/β-catenin pathway factors ,7,8. AmoPrevious reports have demonstrated that Wnt/β-catenin signaling pathways are significantly upregulated in oral cancer cells and malignant oral lesions ,14,15,1622.1The specifications of the SCC-55 cell line used in this study are as follows: site – oral squamous cell carcinoma; region – mandibular, recurrence type of tumor; tumor grade – III. As a control, we have used healthy (non-cancerous) oral squamous epithelial cells obtained in the mandibular region, and no lymphatic nodes are affected. Control cells are named HS-129 and, hereafter, throughout this article, the HS-129 cell line is referred to as “control.” Both SC-55 and HS-129 cell lines are maintained in our Huzhou Central Hospital cell culture depository. These cell lines were cultured in DMEM  containing fetal bovine serum (10%) and antibiotics such as penicillin and streptomycin. The cell culturing conditions/parameters, passaging, and cryopreservation were performed as described previously .2.22. Study groups were assigned as (a) SCC-55 (non-transfected which acts as control); (b) SCC55 + scramble RNAi; and (c) SCC-55 + β-catenin RNAi. Cells at the logarithmic growth phase were subjected to RNAi transfection, mediated by transfection reagent 6 μL of Lipofectamine® 2000 (Invitrogen). The transfection procedure was performed exactly as described in the manufacturer’s protocol. The targeting siRNA for the β-catenin gene (CATGUGUTGGUAAGCUCUA) and scramble RNAi sequence (AUGCUGATCAGUGUCGATU) were used as described previously [SCC-55 cells were grown on six well-plates for 24 h in DMEM with 10% FBS at 37°C with a supply of 5% COeviously , and the2.3AXIN2 and DKK1 used in this study were as described previously [OCT-4, BMI-1, and GAPDH were described previously [Complementary DNA was prepared from the extracted total RNA using the Fermentas Reverse Transcriptase kit. Subsequently, 1 μL of reverse transcription reaction was subjected to qPCR (Bio-Rad-iCycler) with a total volume of 30 μL reaction with Biorad IQ supermix (Syber Green). The primer sequences for eviously ,19. The eviously ,21. OCT-2.4® 2000. Cell lysates were prepared from 24 h post-transfected cells, and the luciferase activity was determined according to the manufacturer’s protocol .Cells were grown for 24 h, subjected to transfection with a reaction mix containing TOPFLASH/FOPFLASH of 75 ng and pCMV-RL (20 ng), mediated by Lipofectamine2.5Total cellular proteins were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Subsequently, proteins were transferred to nitrocellulose membranes and blocked with a 5% skim milk solution in TBST. Then, they were incubated with the primary antibodies such as cytochrome c ; caspase 3 ; caspases 9 ; BCL-2  and GAPDH ; Axin2 (1:500); DKK1  and β-catenin  from Thermofishers. The HRP conjugated secondary antibody, either mouse or rabbit conjugated, was purchased from cell signaling . The protein signal was determined with the ECL chemiluminescence kit (Biorad ECL).2.6Propidium iodide staining (Sigma-Aldrich Kit) was performed in 48 h post-transfected siRNA cells. After overnight incubation at 37°C in dark conditions, cells were fixed in 70% ice-cold methanol and the apoptosis rate was evaluated by flow cytometry. The apoptosis percentage was measured by the mean intensity of the fluorescence signal emitted by dead cells. The mean values represented in the quantification graph were obtained from three individual experiments.2.76) were seeded in 96 well plates, cultured, and the proliferation rate at OD 450 nm was measured from day 1 to day 7. Before each measurement, CCK-8 solution (10 μL) was added to the culture and incubated for 3 h. Thus, the growth curve represents the average OD values of three independent experiments.Cells  × 100.Cells were seeded in 96 well plates and grown for 24 h. Then they were treated with 5-fluorouracil  and cisplatin (20 μmol/L). After 48 h, 10 μL of CCK-8 solution was added to wells, incubated for 3 h, and then chemoresistance assay was performed. The resistance was evaluated precisely using the formula as described previously : rate of2.94 cells were seeded in the top layer and incubated for 2–3 weeks at 37°C. Subsequently, crystal violet staining was performed to visualize and count the colonies.The six-well plates were made of two types of agar layers: bottom layer – 6% agar in DMEM with 10% FBS; top layer – 0.3% agar in DMEM with 10% FBS. Upon solidification, approximately 1 × 102.10The statistical software package was used to perform statistical analysis. The values represented in the error bars are ± standard deviation (SD). In addition, for statistical comparisons between two groups, a one-way analysis of variance (ANOVA) was performed.33.1β-catenin (>7-fold), DKK1 (>5-fold), and AXIN2 (>7-fold) than control cells  SCC-55 cells, we have examined the expression pattern of Wnt/β-catenin genes and its downstream targets. As a result, by western blot analysis, the protein expression level of β-catenin and the downstream targets such as DKK1 and Axin2 are highly upregulated in oral cancer SCC-55 cells compared to control cells . Analogool cells . Conclusol cells . Therefo3.2As Wnt/β-catenin signaling is highly activated in SCC-55 cells, we attempted to knock out β-catenin by RNA interference approach. As a result, our western blot analysis demonstrated that β-catenin expression was dramatically reduced upon β-catenin–siRNA transfection in SCC-55 cells when compared to scramble RNAi and non-transfected (control) SCC-55 cells . Cancer 3.3In vitro proliferation assay demonstrated that the proliferation rate of β-catenin-depleted SCC-55 cells was significantly deteriorated from day 1 to day 7 than the non-transfected and negative control ones (Next, we have examined whether the β-catenin downregulation has a deleterious effect on the tumorigenic potential of oral cancer cells. rol ones . Also, trol ones . Hence, 3.4OCT-4 gene was accelerated in β-catenin–siRNA cells (OCT-4.It has been well demonstrated that cytochrome c release is crucial for the onset of mitochondrial-mediated intrinsic apoptosis through the activation of caspases. Consequently, the protein expression pattern of cytochrome c, caspases 3 and 9 were evaluated by western blot analysis, and they were all found to be highly upregulated in β-catenin–siRNA cells . ConcurrNA cells , compare4In recent times, tumor initiation, progression, and invasion have become complex mechanisms that involved many cell adhesion molecules and intracellular signaling pathways for cell migration and invasion ,24. Mainβ-catenin is an essential stimulation center for Wnt signaling pathways; cytoplasmic multifunctional protein can interact with E-cadherin adhesion molecule in a calcium-dependent manner ,31. OverIn the present article, we have used siRNA that could specifically target β-catenin and suppress its expression. Our RT-PCR and western blot data confirmed the significantly downregulated levels of β-catenin, and thus, the siRNA used could effectively inhibit the Wnt signaling pathway. Interestingly, our flow cytometry analysis demonstrated that apoptosis was significantly initiated in β-catenin-depleted SCC-55 cells. As a result, these cells became more susceptible when treated with DNA targeting drugs such as 5-FU and cisplatin. The precise molecular mechanism of apoptosis induction in SCC-55 cells upon β-catenin gene depletion is not known. However, we found elevated caspase-3 and caspase-9 in β-catenin RNAi cells, indicating that PI3/AKT signaling might be downregulated, promoting mitochondria-mediated intrinsic apoptotic pathways ,31,32,33OCT-4 gene transcription was significantly downregulated in SCC-55 cells upon β-catenin silencing. Elevated expression of OCT-4 has been identified in several cancers, and it acts as a critical regulator of cancer invasion and colony formation [OCT-4 results in decreased tumor invasion and colony formation efficiency in lung cancer [The present study has also demonstrated that the cell proliferation rate slowed down dramatically, and the number of colonies formed in the soft agar assay was significantly compromised when the β-catenin gene expression was downregulated. By considering these data, it can be suggested that the silencing of β-catenin may directly disrupt the expression of genes involved in the regulation of the cell cycle, cell proliferation, and self-renewal of oral cancer cells. Accordingly, our RT-PCR analysis revealed that ormation ,35,36. FOCT-4 [OCT-4. However, further research is required for elucidating the β-catenin-mediated anti-apoptotic mechanism, and its downstream effectors would undoubtedly provide more insights for precisely disrupting the cascade events of Wnt signaling pathways to kill the cancer cells.On the other hand, it has been reported that the therapeutic drug resistance of liver cancer cells is mediated by overexpression of OCT-4 ,36. Ther


Pi enters cells from the extracellular fluid through phosphate transporters and may be stored as PolyP. Here we show that high extracellular Pi concentration in vitro increases platelet PolyP content, in a manner dependent on phosphate transporters, IP6K and V-type ATPases. The increased PolyP also enhanced PolyP-dependent coagulation in platelet-rich plasma. These data suggest a mechanistic link between hyperphosphatemia, PolyP and enhanced coagulation, which may be important in pathologies such as chronic kidney disease.Platelet-derived extracellular polyphosphate (PolyP) is a major mediator of thrombosis. PolyP is a linear chain of inorganic phosphate (P Platelet-derived extracellular polyphosphate (PolyP) is a major mediator of hemostasis, thrombosis, and vascular inflammation and is a promising therapeutic target . Platelei) found in both prokaryotic and eukaryotic cells. In bacteria, intracellular PolyP is involved in energy storage, protection from heavy metal toxicity, and as a molecular chaperone[i concentration ([Pi]cyt). Since Pi is essential to signal transduction and energy metabolism, its levels in the cytosol must be tightly controlled. For example, endothelial cells undergo plasma membrane blebbing and release microparticles when challenged with high [Pi]ex (hyperphosphatemia) can be caused acutely by excessive phosphate intake or chronically by insufficient renal excretion and contributes to the high cardiovascular risk of chronic kidney disease (CKD), which includes vascular calcification and abnormal coagulation[i]cyt, since Pi enters cells from the extracellular fluid through phosphate transportersex) on platelets, and specifically whether this affects platelet PolyP levels.Normal plasma ; and 2.5, which occurs in hyperphosphatemia[i-loading in the presence or absence of TRAP-6 was measured as previously described[i]ex in PRP showed variation between samples, though in none was [Pi]ex greater than 1.1 mM. Where [Pi]ex was lower than 1 mM, it was increased to 1 mM by addition of NaH2PO4. Hyperphosphatemia was mimicked by addition of NaH2PO4 to give [Pi]ex of 2.5 mM.Washed platelets were isolated from blood drawn from healthy drug-free volunteers with approval from the Human Biology Research Ethics Committee, University of Cambridge, as previously described, except sphatemia. PolyP wsphatemia. DNA or sphatemia. This fldescribed. [Pi]ex i]ex (2.5 mM)  . This ef2PO4 was added to increase [Pi]ex, using the malachite green phosphate assay titrate the NaH2PO4 required to elevate [Pi]ex to 2.5 mM. After 90 minutes, coagulation was initiated by addition of CaCl2 (20 mM). Clot turbidity was measured absorbance (405 nm) in a microplate reader without shaking . CaCl2-triggered clotting under these conditions (no exogenous tissue factor) was dependent on PolyP as it was abolished by the addition of alkaline phosphatase  was prepared from blood drawn into sodium citrate (3.2%). NaHsphatase . There wo 2.5 mM . Togethei must enter platelets to increase PolyP levels, we used phosphonoformic acid  to block phosphate transporters. Pre-treatment with PFA prevented the increase in PolyP in response to high [Pi]ex  have been previously implicated in PolyP synthesis and storage in platelets ,13. The of IP6K1 .i]ex. We propose that Pi enters platelets through phosphate transporters and is converted to PolyP and stored in dense granules in a mechanism dependent on IP6K and V-type ATPases . Although the concentration used in our study is high, it is possible that similar concentrations can be achieved in patients, althougTrypanosoma and Leishmania, PolyP synthesis requires a vacuolar transporter chaperone complex. PolyP that is synthesized at the cytoplasmic face of acidocalcisome-like organelles by this complex is translocated into the organelle lumen. A transmembrane proton gradient generated by V-type ATPase activity is also requiredex was inhibited by TNP.IP6K1 also regulates PolyP levels in eukaryotic cells. Although normal function of IP6K1 is to synthesis 5-diphosphoinositol pentakisphosphate (IPplatelets. Plasma It would be interesting to investigate whether platelets from patients with hyperphosphatemia may have increased PolyP levels and whether this contributes to abnormal coagulation. Since it is possible to block the increase in PolyP levels, as suggested by our data, or the pro-thrombotic effects of PolyP, PolyP i


Interest in CBG (cannabigerol) has been growing in the past few years, due to its anti-inflammatory properties and other therapeutic benefits. Here we report the synthesis of three new CBG derivatives  and show them to possess anti-inflammatory and analgesic properties. In addition, unlike CBG, HUM-234 also prevents obesity in mice fed a high-fat diet (HFD). The metabolic state of the treated mice on HFD is significantly better than that of vehicle-treated mice, and their liver slices show significantly less steatosis than untreated HFD or CBG-treated ones from HFD mice. We believe that HUM-223, HUM-233 and HUM-234 have the potential for development as novel drug candidates for the treatment of inflammatory conditions, and in the case of HUM-234, potentially for obesity where there is a huge unmet need. CBG (cannabigerol) was discovered by Gaoni and Mechoulam in cannabis resin (hashish) in 1964 and was considered a missing link in the biosynthesis of THC (tetrahydrocannabinol) .While the cannabis constituents CBD and THC have been thoroughly investigated ,4, reseaIndeed, CBG has been shown to exert anti-inflammatory effects and some derivatives of CBG have been synthesized and tested in both animal models and human patient ,6,7,8,9.The immune and inflammatory system has evolved to protect against foreign organisms and substances, antigens, that may cause damage to the normal function of the body. The immune response, when dysregulated, may also lead to various pathological conditions, including tumors , autoimmHere we report the synthesis of three new synthetic CBG derivatives and show them to possess anti-inflammatory and pain-resolving properties in preclinical models. In addition, one of these molecules, HUM-234, has also shown prominent activity in obesity prevention in mice fed a high-fat diet (HFD). Importantly, the metabolic state of the HUM-234 treated HFD mice is significantly closer to healthy levels than that of vehicle-treated HFD mice, and their liver slices show much less steatosis than untreated, or CBG-treated livers from HFD mic, suggesting that HUM-234 and related compounds may have potential to treat metabolic disorders including liver disease.4 (Lithium Aluminium Hydride) to produce the final HUM-223.We sought to explore the effect of a bulky residue at position 5 of CBG in place of the natural pentyl on its anti-inflammatory activity. This is based on similar observations made for THC, among other cannabinoids, in which a bulkier residue such as dimethylheptyl (DMH) resulted in an improved efficacy at the relevant biological model . We haveAnother alteration deemed to be useful, namely the use of a morpholine propionate ester at one of the phenol positions of CBG. This concept is based on observations made by our group for a similar modification of CBD, which produced an improvement in the anti-inflammatory properties . CouplinTo further improve its bioavailability, HUM-234 was also turned into a maleic acid salt named HUM-233 . This waHUM-223 was compared to CBG and to a vehicle control group in three inflammation assays: paw swelling, pain sensation in the paw and circulating TNF-α . CBG itsThe anti-inflammatory ability of HUM-223 was also assessed in zymosan-induced arthritis (ZIA), a model of acute inflammation in the knee , in compThe biological results revealed that both HUM-233 and HUM-234 have anti-inflammatory activity . Both coDespite having comparable anti-inflammatory activity, HUM-234 is much more active than CBG in the prevention of obesity. Adult female mice fed an HFD (high-fat diet) gradually gain weight, much faster than STD (standard diet) fed ones. CBG  does not limit weight gain in our model. On the contrary, the weight gain is at some time points even higher for this group than for the vehicle-treated HFD group. However, HUM-234-treated mice (25 mg/kg) gain weight much slower than HFD or HFD + CBG groups . The ALSIt is of great interest that while the anti-inflammatory and pain-relieving activity of CBG and HUM-234 are similar, HUM-234 ameliorates weight gain, in contrast to CBG, suggesting this compound has the potential for development as an anti-obesity drug.Here we report the synthesis of, and the evaluation in-vivo of the anti-inflammatory, analgesic and anti-obesity of three novel CBG derivatives.Inflammation contributes to the pathogenesis of many diseases. Chronic inflammation can lead to cardiovascular diseases, gastrointestinal diseases, obesity, asthma, arthritis, neurodegenerative diseases, cancer and more.Currently, the drugs used to treat inflammation are most often NSAIDs and steroids. Monoclonal antibodies to cytokines, TNF, IL6 and IL12/23 are used if these do not work. NSAIDs work by inhibiting the activity of cyclooxygenase enzymes (COX-1 or COX-2). In cells, these enzymes are involved in the synthesis of prostaglandins, which are associated with inflammation, and thromboxanes, which are involved in blood clotting . Most NSOf equal if not greater concern are side effects associated with long-term use of steroidal drugs. Although highly effective in the elimination of inflammation, they can cause obesity, growth retardation in children, and even lead to convulsions and psychiatric disturbances, osteoporosis, adrenal suppression, hyperglycemia, dyslipidemia, cardiovascular disease, Cushing’s syndrome, immunosuppression. an increase in the rate of infections .There has been progress in the treatment of many inflammatory diseases, initially rheumatoid arthritis, and Crohn’s disease, then many others. TNF (Tumor Necrosis Factor) inhibitors have been an important step forward in the treatment of several chronic inflammatory diseases, especially rheumatoid arthritis, and Crohn’s disease. But these drugs are injectable and costly and have a potential for adverse effects, such as reactivation of latent tuberculosis. Furthermore, many patients treated with TNF inhibitors continue to experience chronic pain and so there is a big unmet need for cheaper, orally available drugs that reduce inflammation and pain .The search for easily prepared, small molecular weight compounds, which can be delivered as an oral formulation continues. A hitherto mostly untapped wealth of novel compounds are natural products from plants, known to possess anti-inflammatory properties. Indeed, Cannabis sativa preparations and pure cannabinoids have been used to relieve symptoms of pain associated with several clinical disease conditions and ailments.Previously we have reported the inflammatory disease modulating properties of CBD in several in vitro and in vivo systems and have demonstrated a potent ameliorating effect on the clinical signs of arthritis in a model of Rheumatoid Arthritis (RA) . In a reThe synthesis of HUM-223, HUM-233 and HUM-234 uses CBG as starting material. CBG is formed in the plant (or by heating) from its precursor CBGA . CBG aciThe in-vivo assays used in the present study are well established and widely used in inflammation research ,42. The HUM-223 was able to significantly reduce swelling in all doses and exceed CBG’s effect at a dose of 10 mg/kg. In addition, it showed activity in reducing pain responses and reducing TNF-α levels. The latter effects were comparable to CBG.We chose HUM-223 to be tested on the knee-arthritis model to complement the described paw arthritis model, as previously similar CBD derivatives, possessing DMH (dimethylheptyl) side chain were more active than pentyl chain derivatives in this model (unpublished results). We were able to prove that HUM-223 is indeed efficacious and doses of 5, 10 and 25 mg/kg have a comparable response to dexamethasone. A dose of 5 mg/kg HUM-223 was proved as the most effective dose out of the three doses tested. At this dose, the effect of HUM-223 was similar to dexamethasone, a known highly effective anti-inflammatory steroidal drug. Furthermore, HUM-223 was shown to reduce local gene expression of Elane, Mpo and Adamts4 at the endpoint of the experiment; enzymes upregulated in inflammatory arthritis, shown to play a central role in oxidant production, cartilage degradation and joint damage ,44,45. IRegarding HUM-233 and HUM-234, both showed anti-inflammatory activity comparable to CBG in all three assays. They were all able to reduce swelling, decrease evoked pain responses upon application of localized pressure on the inflamed paw. We observed an inverse dose-response trend upon increasing the dose of the compounds . The salOf notable interest is the effect on weight gain in an established standard model of obesity. We chose HUM-234 to be assayed on diet-induced obesity, as we previously established a similar derivative of CBD, HU-435, could prevent weight gain in mice . Indeed,The effect of HUM-234 on diet-induced obesity is of great interest to us. Obesity is an “epidemic” of the developed world . It causWe are aware of the limitations posed by the compounds’ biological behavior. All three compounds showed some variability in the concentrations most active in the swelling, pain sensation and TNF-α assays as did CBG. This is a known and well-documented trend in cannabis studies . MoreoveWe believe that CBG derivatives HUM-223, HUM-233 and HUM-234 have the potential to be further developed as novel drug candidates for use in inflammatory conditions, and potentially also as anti-obesity treatment, where there is a huge unmet need.NMR data were collected on Varian Unity Inova 300 MHz spectrometer using the standard pulse sequences and processed with Agilent software.The samples were analyzed by GC-MS in a Hewlett-Packard G1800 A GCD system with HP-5971 gas chromatograph with electron ionization detector. Ultra-low-bleed 5%-phenyl capillary column (28 mm × 0.25 mm (i.d.) × 0.25 μm film thickness) based on diphenyl methylsiloxane chemistry  was used. Experimental conditions were: inlet, 250 °C; detector, 280 °C; splitless injection time; initial temperature, 90 °C; initial time, 3.00 min; rate, 25 °C/min; final temperature, 280 °C; helium flow rate, 1.0 mL/min. The software used was GCD Plus ChemStation.® CSH, 2.1 × 100 mm, 2.5 um, Waters (TC) Israel Ltd., Petah Tikva, Israel) connected to Waters 2489 UV/visible Detector and Waters QDa Detector. The Software used was MassLynx V4.2. Several gradients of Acetonitrile and water both containing 0.1% FA were developed for analysis starting from 0% to 100% Acetonitrile or 50% to 100% Acetonitrile. Eventually, the addition of Methanol to the gradient was proved effective for the analysis of our compounds and the final method of analysis was as follows: column temperature: 45 °C, UV detector: 225 nm, sampling rate 20 points/s; QDa detector: ES (+) m/z between 100 to 1000, cone voltage 2V, ES (−) m/z between 100 to 1000, cone voltage 2 V. Gradient: starting point: 15% acetonitrile (0.1% FA), 15% methanol (0.1% FA) and 70% water (5% acetonitrile and 0.1% FA) from 0 to 15 min. Then, 40% acetonitrile (0.1% FA), 40% methanol (0.1% FA) and 20% water (5% acetonitrile and 0.1% FA) from 15 to 22 min. Equilibration to starting conditions from 22 to 27 min. Flow rate: 0.25 mL/min. Probe temperature: 600 °C, source temperature: 120 °C, turbo temperature: 49 °C.LC-ESI-MS was done with Waters LC e2695 Separation Module equipped with reversed phase C18 column . CBG-DMH is purified by repeated chromatography. CBG-DMH is obtained as a pale-yellow oil. Yield: 0.09 gr (30%). Analytical characteristics were in accordance with previously published literature , 494 [M + Na] tR: 7.26 min, ES (−) m/z 115 [M − H], 231 [2M − H] tR: 1.91 min.0.343 gr (0.72 mmol) of HUM-234 are dissolved in 40 mL of isopropyl alcohol (IPA). Then, 83.5 mg (0.72 mmol) of maleic acid are added and the reaction is stirred at room temperature for 2.5 h. IPA is evaporated and HUM-233 is recrystallized from EtOAc and ether. HUM-233 is obtained as crystalline white solid. Yield: 0.32 gr (75%). 1H NMR  are added, followed by 0.04 gr of pyrrolidinopyridine. The reaction is then stirred at room temperature for 5 min. 0.56 gr of N,N-dicyclohexylcarbodiimide (DCC) are added and the reaction is stirred at room temperature overnight. The solids are filtered, and the solution is evaporated. The crude product is purified by silica gel column chromatography (TLC 20% EtOAc:Pet Ether). HUM-234 is obtained as yellow oil. Yield: 0.25 gr (20%). 1H NMR  δ 6.57 , 6.50 , 5.19–4.99 , 3.80 , 3.72 , 3.25 , 2.88–2.66 , 2.55–2.48 , 2.11–2.00 , 1.99–1.89 , 1.73 , 1.65 , 1.57 , 1.39–1.24 , 0.91 . 13C NMR  δ 170.89, 158.12, 149.31, 145.98, 141.99, 134.79, 131.18, 124.32, 122.26, 119.72, 114.34, 108.53, 66.91, 55.70, 54.03, 53.42, 39.75, 35.96, 32.30, 31.57, 30.92, 26.69, 25.71, 22.85, 22.57, 17.69, 16.11, d14.08. LCMS: ES (+) m/z 472 [M + H], 494 [M + Na] tR: 7.26 min.0.432 mL of methyl morpholinoproprionate are dissolved in 4 mL of 1,4-dioxane, 2.16 mL of water and 1.08 mL of 3N aqueous sodium hydroxide. The reaction is stirred at room temperature for 4.5 h and then the solvent is evaporated. The solids are suspended in 40 mL of dry DCM under nitrogen atmosphere and small amount of MgSOad libitum. The mice were acclimatized in the animal facility for at least 2 weeks before the experiments. The data presented in Figures are representatives of 2 separate experiments.Female Sabra mice  and C57Bl6 , 7–8 weeks old, were maintained in the specific-pathogen-free unit of the Hadassah Medical School, Hebrew University, Jerusalem, Israel. The experimental protocols were approved by the Institutional Animal Care Ethics Committee (permission # MD-20-16042-5). The animals were maintained at a constant temperature (20–21 °C) and a 12-h light/12-h dark cycle and were provided a standard pellet diet with water w/v zymosan A  in saline into the subplantar surface of the right hind paw of the mice. This was followed immediately by an intraperitoneal injection of the test compound. For injection, the compounds were dissolved in a vehicle containing ethanol:Cremophore:saline at a ratio of 1:1:18. Paw swelling and pain perception were assessed after 2, 6 and 24 h. Blood was collected after 24 h for analysis of TNFα serum levels.Inflammation was induced by injection of 40 uL of a suspension of 1.5% Calibrated calipers were used to measure paw swelling (thickness) 2, 6 and 24 h after injection of zymosan.Pain at 2, 6 and 24 h after zymosan injection was assessed by the von Frey nociceptive filament assay, where 1.4–60 g filaments, corresponding to 4.17–5.88 log of force, was used to test the sensitivity of the swollen paw. The untreated hind paw served as a control. The measurements were performed in a quiet room and the animals were handled for 10 s before the test. A trained investigator then applied the filament, poking the middle of the hind paw to provoke a flexion reflex, followed by a clear finch response after paw withdrawal. Filaments of increasing size were each applied for about 3–4 s. The mechanical threshold force in grams was defined as the lowest force required to obtain a paw retraction response.Blood was collected 24 h after zymosan injection, and the sera were assayed for TNFα using a mouse TNFα ELISA kit , according to the manufacturer’s instructions.All experimental procedures were approved by the Ethical Review Process Committee and the UK Home Office, in accordance with the 1986 Animals (Scientific Procedures) Act (permission # 30/3441). Male C57BL/6J mice aged 8–10 weeks were used. Mice were housed in ventilated cages, maintained at 21 °C ± 2 °C and a 12-h light/12-h dark cycle, with food and water available ad libitum. Mice were anesthetized with 2% isoflurane and both knees were shaved. ZIA was induced by intra-articular injection of 180 µg of Zymosan A (Sigma) suspended in PBS as previously described . Images Mice were fed with a standard diet (STD) or high-fat diet (HFD) for 7 days before the beginning of the injections. On day 7 they were divided into the groups: STD, HFD, HFD+CBG and HFD+HUM-234, and treated for 35 days. The mice were weighed every week.At the end of the diet-induced obesity experiment, the mice were sacrificed. The livers were removed, fixed with buffer formalin and stained with haematoxylin and eosin, for microscope evaluation. Paraffin sections of 4–5 µm thickness, were stained with hematoxylin and eosin. For microscopic evaluation, sections were examined (×40).The levels of two aminotransferases, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were assayed in the sera of mice at the end of diet-induced obesity experiment with or without HUM-234 treatment, by ALT and AST strips respectively  and quantitated by an automated analyzer .p-value < 0.05, ** p-value < 0.01 and *** p-value < 0.001 when comparing to a control group. # p-value < 0.05 when comparing between the different dosages of the tested compounds, or when comparing them to CBG, the exact comparisons are listed on Figures’ legends.Statistical analysis was performed with GraphPad Prism software. Statistical analysis details are listed under each figure. The results are presented as value ± SE (standard error). In rare cases where all the measurements give the same values, no SE bar is presented, as no error can be measured. *


The results show that ionic space charge accumulation was significantly suppressed in the composite at high temperatures. The conduction current increased gradually with temperature and manifested a remarkable shift from electron charge transport to ion charge transport near the glass transition temperature (Tg). The real and imaginary permittivity showed an enormous increase above Tg for both the epoxy resin and the composite. The conduction current and permittivity of the composite were remarkably reduced in comparison to the epoxy resin. Therefore, the ionic process dominated the high temperature dielectric properties of the epoxy resin and the composite. The nano–micro fillers in the composite can significantly inhibit ion transport and accumulation, which can significantly enhance the dielectric properties of epoxy resin. Thus, the nano–micro composite has a strong potential application as a package material and insulation material for electronic devices and electrical equipment operated at high temperatures.Thermally conductive epoxy resin composites are widely used as electrical equipment insulation and package materials to enhance heat dissipation. It is important to explore the dielectric properties of the composites at high temperatures for the safe operation of the equipment. This paper investigated the charge transport behavior of an epoxy/nano−SiO With improvements in the integration degree and power density of electrical and electronic equipment, the heat generated per unit volume of equipment increases significantly. The continuous heat accumulation and the resulting temperature rise will accelerate the aging of the insulating dielectric and greatly reduce the reliability and life of the electrical and electronic equipment ,2. ThereEpoxy resin is widely used in electrical equipment insulation systems and electronic device packaging due to its excellent adhesion, corrosion resistance and dielectric properties ,5,6,7,8.Additionally, the thermal conductivity of epoxy resin is only about 0.2 W/(m·K), which seriously affects the heat dissipation performance of the device. Some studies have shown that doping a certain amount of thermal conductive fillers in polymers can improve the thermal properties but adding too much filler will cause poor dielectric properties of the materials ,13. Ther2/micro−BN composite at varied temperatures is investigated by combined analysis of the TSDC , conduction current, complex permittivity and space charge distribution between 40 and 200 °C.In this paper, the charge transport behavior of an epoxy/nano−SiO2 and BN particles. SiO2 was provided by Denka, Japan, which had an average particle size of 15 nm added to a ratio of 4 wt%. BN was purchased from Aerosil, Japan, in the shape of a flake with a diameter of 8 μm (D50), and was added to a volume ratio of 10 vol%. Volume ratio has a significant effect on improving thermal conductivity, so we used vol% for the ratio of the micro filler. Epoxy resin (diglycidyl ether of bisphenol−816B) was obtained from Mitsubishi Chemical . The weight ratio of curing agent  to epoxy resin was 30 to 100. The fillers included SiO2 and BN fillers in a planetary centrifugal mixer  for 3 min. Second, add the filler mixture to the epoxy resin and mix in the same mixer for 10 min. Third, evacuate the mixture in a vacuum oven at room temperature for 30 min in order to degas sufficiently. Fourth, mix the sufficiently degassed mixture with the hardener added into it for five minutes. Fifth, pour the previous mixture into a stainless steel mold and press tightly. Finally, precure it at 70 °C for three hours and postcure it at 120 °C for another three hours. Then, the sample obtained is naturally cooled down to room temperature. Hereafter, the thickness of the sample is 150–300 μm. The composite was prepared with the following procedure. First, mix SiOScanning electronic microscopy (SEM): The dispersion of micro−sized and nano−sized fillers in the epoxy composite was studied using a thermal field emission scanning electron microscope  to observe the cross−section of the sample after extraction with liquid nitrogen. The test voltage was 10 kV.−2 to 106 Hz. Complex permittivity measurement: The dielectric properties were measured using an impedance analyzer . Samples of 200 μm thickness were operated at an AC voltage of 3 Vrms, at temperatures from 20 to 200 °C and at a frequency range from 10Conduction current measurement: The conduction current was measured using a Digital electrometer (Advantest R8252). The samples were operated in an atmosphere of nitrogen, at temperatures from 40 to 200 °C; the test voltage was a DC electric field (30 kV/mm). Space charge distribution measurement: The conducted currents under short−circuit, positive and negative electric field conditions were measured using a pulsed electroacoustic (PEA) device  to study the space charge distribution of samples. The electric field was 10 kV/mm. Thermally stimulated discharge current measurement (TSDC): The Digital electrometer (Advantest R8252) was used to measure the thermally stimulated discharge current from 40 to 200 °C with a heating rate of 2 °C/min. 2/micro−BN composite were used in all the tests to ensure the repeatability of the experimental results.At least two different samples of both the epoxy resin and the epoxy/nano−SiO2 nanoparticles in the epoxy/nano−SiO2/micro−BN composite is shown in the SEM image in The dispersion of the micro BN particles and SiOThe spatial charge distribution in the epoxy resin polarized at test temperatures of 40 °C, 60 °C, 80 °C and 100 °C is shown in 3. The space charge distributions in EP polarized at temperatures of 120, 140, 160, 180 and 200 °C are shown in 2/micro−BN composites polarized at temperatures from 40 to 200 °C are shown in Tg) of epoxy resin and the composite is between 100 and 120 °C, and the increase in heterocharges becomes much less significant when the temperature is above 100 °C. The homocharges injected by the electrode are just hidden by heterocharges above 120 °C [The space charge distributions in epoxy/nano−SiOe 120 °C .2/micro−BN composite as a function of temperature from 40 to 200 °C. Among them, the ion density of the composite remains below 10 C/m3 all the time. However, the accumulated ionic space charges in epoxy resin are greater than those in the epoxy composite above 100 °C. Therefore, SiO2–BN fillers can significantly inhibit ion transport and accumulation in the epoxy/nano−SiO2/micro−BN composite.2/micro−BN composite with time at different temperatures varies, as shown in 2/micro−BN composite changes over time at varied temperatures between 20 and 200 °C. From The conduction current behavior of epoxy resin and the epoxy/nano−SiOEA) of both epoxy resin and the epoxy/nano−SiO2/micro−BN composite is higher at 120–200 °C than 40–100 °C. The activation energy of the epoxy composite (0.9 eV) is lower than that of pure epoxy (1.20 eV) at a high temperature. The mobility of polymer molecular chains at the interface area in the composite is inhibited due to the steric hindrance effect of nano− and micro fillers, which equally introduces deep trapping sites at the interface area; thus, ionic charge transport in the composite is suppressed compared to the pure polymer [Ions are transported through thermally activated hopping with a mobility that obeys the Arrhenius equation  and the imaginary part (εr″) of the complex permittivity of epoxy resin and the composite increase significantly with temperature. This shows that at a higher temperature and lower frequency, ions move to the electrode interface, and the polarization of the electrode induces a large amount of mirror charge, which causes a large number of ions to gather around the electrode; thus the εr′ and εr″ increase to high values. Furthermore, it has verified that the glass transition temperature is about 100 °C, and the increase in εr′ is due to the accumulation of impurity ions near the electrode. When the test temperature of epoxy and the composite material is higher than the glass transition temperature, the molecular chain movement in the amorphous region of the material is more active, which significantly improves the charge transfer ability. The charge can easily transfer to the other side of the electrode, forming electrode polarization. However, due to the barrier between the electrode and dielectric interface, charge extraction is limited and heteropolar space charge is formed. The higher the temperature of heteropolar space charge is, the more ions are activated. Therefore, electrode polarization and ion charge transport at high temperatures can make the complex dielectric constant of the material larger. There are two main reasons for the generation of ions in epoxy resin: first, a small amount of sodium ions and chloride ions remain in the synthesis process of epoxy resin in an alkaline environment; second, polar impurities and unreacted parts of the curing agent are ionized at high temperatures. An obvious relaxation process occurs in the spectra when the ambient temperature of epoxy and the composite is lower than the glass transition temperature. It is mainly because the relaxation process is triggered by the orientation of molecular chain side chains and side groups, and the relaxation process moves to a higher frequency with temperature. r′ and εr″ of epoxy and the composite increase significantly with the temperature above the glass transition temperature, and compared with the epoxy resin, the εr′ and εr″ of the composite are lower. This is because the nano and micro fillers in the composite material fill the gap between the molecular chains and hinder the movement of the molecular chains, resulting in ion transport becoming difficult when the test temperature is above Tg. This conclusion is further confirmed by the fact that the SiO2–BN fillers can significantly inhibit ion transport and accumulation in the epoxy composite at high temperatures.The changes in the complex dielectric constants of epoxy resin and the composite at 0.01 and 0.1 Hz with temperature are shown in 2/micro−BN composite as a function of temperature from 20 to 200 °C. By comparing the TSDC spectra of epoxy resin and the composite, it can be known that the current peak in both materials appears at 80–110 °C. Furthermore, when the temperature is above 140 °C, the current peak is ascribed to the ion transport.2/micro−BN composite. For epoxy resin, the peak current increases from 80 to 120 °C, reaches the maximum at 120 and 140 °C and then decreases from 140 to 200 °C. For the epoxy composite, the peak current values are nearly the same for all polarization temperatures, and the current peak moves to a high temperature with the increase in Tp, indicating a dispersion of the α relaxation process. In addition, both the peak current and peak temperature of the epoxy composite are higher than those of epoxy resin, indicating that it is more difficult for ions to transport in the composite, which is also confirmed by the conductivity.2/micro−BN composite under varied temperatures have been investigated. The results suggest that the space charge behavior in epoxy resin and the composite is dominated by electronic charge accumulation below the glass transition temperature (Tg) and by ion accumulation at temperatures above the Tg. The enormous increase in the complex dielectric permittivity of both epoxy resin and the composite is due to electrode polarization caused by ion charge transport above the Tg. However, SiO2–BN nano–micro fillers can significantly inhibit ion transport and accumulation by hindering the movement of the molecular chains, thus leading to a lower conduction current and higher ρ peak temperature in TSDC spectra.The space charge distribution, conduction current, complex permittivity and TSDC of epoxy resin and an epoxy/nano−SiO2/micro−BN composite exhibits remarkably improved dielectric properties at high temperatures compared to epoxy resin and is more suitable for the insulation materials in equipment and devices operating at high temperatures.In summary, the nano–micro fillers have a steric hindrance effect on epoxy chain segment movement and lead to limited ion transport. The epoxy/nano−SiO