Triple negative breast cancer (TNBC) is characterized as a heterogeneous disease with severe malignancy and high mortality. Aberrant Wnt/β-catenin signaling is responsible for self-renewal and mammosphere generation, metastasis and resistance to apoptosis and chemotherapy in TNBC. Nonetheless, in the absence of a targeted therapy, chemotherapy is regarded as the exclusive treatment strategy for the treatment of TNBC. This review aims to provide an unprecedented overview of the plants and herbal derivatives which repress the progression of TNBC through prohibiting the Wnt/β-catenin pathway. Herbal medicine extracts and bioactive compounds  alone, in combination with each other and/or with chemotherapy agents could interrupt the various steps of Wnt/β-catenin signaling, i.e., WNT, FZD, LRP, GSK3β, Dsh, APC, β-catenin and TCF/LEF. These phytotherapy agents diminish proliferation, metastasis, breast cancer stem cell self-renewal and induce apoptosis in cell and animal models of TNBC through the down-expression of the downstream target genes of Wnt signaling. Some of the herbal derivatives simultaneously impede Wnt/β-catenin signaling and other overactive pathways in triple negative breast cancer, including: mTORC1; ER stress and SATB1 signaling. The herbal remedies and their bioactive ingredients perform essential roles in the treatment of the very fatal TNBC via repression of Wnt/β-catenin signaling. Breast cancer was reported as the most diagnosed cancer and the leading cause of cancer mortality worldwide among women in 2020 . AdvanceThe Wnt signaling commences with the binding of Wnt ligands to the N-terminal extra-cellular cysteine-rich domain of the Frizzled (FZD) seven-pass transmembrane receptors and the LDL receptor-related proteins (LRP coreceptor). The trimetric complex  recruits phosphorylated Disheveled (Dsh) and Axin, to prevent β-catenin phosphorylation. Therefore the APC/CK1/GSK-3β/Axin/β-catenin degradation complex is inactivated and the phosphorylation of β-catenin by GSK-3β is repressed. The accumulated β-catenin in the cytoplasm translocates into the nucleus, where it modulates the target gene expression with the T cell factor/lymphoid enhancer factor (Tcf/Lef) family of transcription factor . BesidesAlthough chemotherapeutic agents have been employed as exclusive treatments of TNBC, the resistance to chemotherapy agents , biomarker-based treatments and checkpoint-based immunotherapy has led to inconsequential responses to the medications. An extensive spectrum of clinical investigations has claimed the favorable influence of herbal remedies on the survival, immune enhancement and quality of life of cancer sufferers . MoreoveRadix bupleuri L. (Chaihu)—a known Chinese traditional medicine with broad applications. The Chinese Food and Drug Administration (CFDA) has approved fifteen clinical preparations of Radix bupleuri -2-methoxyphenol, is a bibenzyl-type phenolic biomolecule extracted from various medicinal orchids, with numerous pharmaceutical benefits . An inveDrynaria fortunei (Kunze) J. Sm., Citrus aurantium L. and Citrus medica L. It occurs in citrus fruit and endows bitterness to the citrus juices [Naringin, a flavanone glycoside composed of the flavanone naringenin and neohesperidose, is a crucial bioactive ingredient of s juices . The ants juices . The nucs juices .Sophoraflavescens Ait, possesses diverse medicinal qualities [Oxymatrine, a quinolizidine alkaloid compound isolated from the roots of ualities . Oxymatrualities . The monualities ,36. Xie ualities .SilybumMarianum L. Gaertn), elucidated chemopreventive and chemo-sensitizing properties against various cancers [Silibinin, an essential bioactive flavonolignan extracted from milk thistle  as a negative transcriptional regulator of Wnt signaling . HBP1 isZingiberaceae family. There is mounting evidence supporting the multi-potency anti-cancer activities of Cardamonin via modulating various signaling pathways, transcriptional factors, cytokines and enzymes, such as mTOR, NF-κB, Akt, STAT3, Wnt/β-catenin and COX-2 [Cardamonin,  is a naturally occurring chalcone, commonly derived from different plants of the nd COX-2 . The brend COX-2 .Inonotus obliquus (Chaga mushroom), an edible fungus which inhabits birch trees [Inonotus obliquus exhibited potent anti-inflammatory and anti-proliferative properties through the downregulation of Wnt/β-catenin [Inotodiol is a natural lanostane-type triterpenoid isolated from ch trees . A previ-catenin . The eff-catenin . It was -catenin . The res-catenin . This fi-catenin .Schisandra chinensis (Turcz.) Baill. [Schisandrin A is the most dominant lignan (polyphenolic compounds) present in the fruit of ) Baill. . The ant) Baill. . The acc) Baill. . TherefoResveratrol  is a plant polyphenolic derivative, widely found in grapes, berries and peanuts . The cliMundulea sericea L., belonging to the Leguminosae family [Deguelin is one of the major naturally occurring rotenoids, extracted from e family ,67. It w+ and mesenchymal–like CD44+/CD24−/low BSCS subpopulations. In addition, it suppressed the migration and invasion of various TNBC cell lines  [Hydroxytyrosol  is a phenolic alcohol extracted from olive oil . Cruz-Lo Hs578T) . These r Hs578T) . Hydroxy Hs578T) .Fucoidan, a polysaccharide obtained from brown seaweed and some marine invertebrates, consists of considerable amounts of L-fucose and sulfate ester groups. It may also contain other monosaccharides  and uronic acids, acetyl, sulfate and protein . It was Euphorbiaceae family, such as Jatropha isabelli and Jatropha gossypiifolia [Jatrophone is a macrocyclic diterpene, consisting of an oxaspiro core and several electrophilic centers. Jatrophone is isolated from the piifolia . Fatima’piifolia .Luteolin, 3,4,5,7-tetrahydroxy flavone, is a natural flavonoid, extensively present in many fruits and vegetables, such as celery, sweet bell peppers, carrots, broccoli, onion leaves and parsley . The antTripterygium wilfordii Hook F. [Triptolide, diterpene triepoxide, a natural component, is extracted from the traditional Chinese medicinal plant  Hook F. . It was  Hook F. .Astragalus membranaceus L., a traditional Chinese remedy [Astragalus polysaccharide on human triple negative breast cancer cells (MDA-MB-231) was evaluated and evidenced by MTT and Ki67 immunofluorescence staining assays. Furthermore, it was found that Astragalus polysaccharide therapy prohibited the migration and invasion of these cells. These findings were consistent with RT-qPCR, Western blot and immunofluorescence staining analysis, as they confirmed the impediment of EMT via the downregulation of the mesenchymal marker Vim and the EMT-related transcription factor Snail, and the upregulation of E-Cad. Following Astragalus polysaccharide exposure, the expression of β-catenin and its downstream oncoproteins c-Myc and Cyclin D1 were reduced. The repressive potential of this hetero-polysaccharide on metastasis was exerted via altering the expression of the Wnt/β-catenin- and EMT-related genes. Moreover, it was demonstrated that lithium chloride (LiCl), an agonist of the Wnt/β-catenin pathway, inverted the suppressive effect of Astragalus polysaccharide on the Wnt/β-catenin pathway [Astragalus polysaccharide, a water-soluble hetero polysaccharide, consisting of α-1,4--glucan, rhamnus-galacturonic acid polysaccharide I, arabinogalactan polysaccharide and arabinogalactan protein polysaccharide. It is an essential bioingredient isolated from the roots of e remedy ,81. The Quercetin  is a yellow pigment bioflavonoid contained in numerous fruits, vegetables, seeds, nuts, green tea and red wine ,83. SrinN-(6-chloro-2-methoxyacridin-9-yl)-1-N,1-N-diethylpentane-1, 4-diamine), is a quinine derivative extracted from the bark of the cinchona tree [Quinacrine . The treatment of cells with Quinacrine followed by Lycopene, decreased the TCF/LEF activity compared to only Quinacrine therapy. The co-treatment with Quinacrine and Lycopene upregulated the APC expression while it downregulated β-catenin and Cyclin D1. Lycopene improved the anti-proliferation potential of Quinacrine by prohibiting the WNT/TCF signaling in the MDA-MB-231 cells .Curcuma longa L. [Curcumin -1,6-heptadiene-3,5-dione) is a yellow-colored natural hydrophobic polyphenol occurring in the rhizome of the longa L. . Prasad’longa L. .In another piece of research on SUM159 cells, it was displayed that the Curcumin treatment reduced the tumor sphere formation and downregulated the BCSCS markers , cell-proliferation proteins (PCNA and Cyclin D1) and the anti-apoptotic protein (Bcl2), and upregulated the apoptotic proteins  . It is wCrocus sativus L. (saffron) [Crocin, 8, 8-diapocarotene-8, 8-dioic acid, is the essential water-soluble carotenoid extracted from the dried stigmas of saffron) ,93. Arzisaffron) ,95.In a complementary piece of research, our group investigated the anti-metastatic efficiency of the combination of Crocin and Crocetin, another major carotenoid of saffron, on the cell and mice models of TNBC. It was shown that the combination therapy of 4T1 cells reduced proliferation, migration, invasion and adhesion to ECM, even more effectively than Crocin and/or Crocetin. The xenograft mice assays confirmed these outcomes, as the combination-treated mice possessed less metastatic deposits in the liver and lung tissues. The Western blot and real-time PCR findings exhibited that the combination therapy reduced the expressions of Fzd7, Nedd9, VEGF-α, Vim and Mmp9 and upregulated E-Cad in 4T1 cells and liver and tumor tissues, thus preventing Wnt/β-catenin signaling and averting metastasis in TNBC .The chemical structures of the natural derivatives combating TNBC through interfering with the Wnt/β-catenin pathway are demonstrated in Rehmannia glutinosa (Gaetn.) Libosch. ex Fisch. et Mey (Shudi Huang); Dioscorea opposita Thunb (Shan Yao); Cornus officinalis Sieb. et Zucc (Shanzhu Yu); Poriacocos (Schw.) Wolf (Fu Ling); Alisma orientalis (Sam) Juzep (ZeXie) and Paeonia suffruticosa Andr (Mudan Pi) [The Liuwei Dihuang pill (LWDHP) is a classic Chinese herbal medicine containing six herbs: udan Pi) . It has udan Pi) ,99. Lixiudan Pi) . Moreoveudan Pi) .Syzygium guineense Wall., a member of the Myrtaceae family, is a widespread plant native to the regions of Australia, Asia and Africa. It has been prescribed as a medicinal plant due to its anti-tumor activity against TNBC cells [S. guineense extract therapy of malignant cells lead to an inhibition of cell proliferation through blocking the Wnt3a-induced β-catenin stabilization and suppression of the Wnt-dependent transcription. It is also noteworthy that phyto-analytical studies have introduced tannins, a group of polyphenols, as an effective inhibitor of Wnt3a-stimulated signaling and TNBC cell proliferation [BC cells ,103. KovGanoderma lucidum (Reishi or Lingzhi), the most common species in the genus Ganoderma, is a famous Chinese traditional medicinal plant, which has found novel applications as a cancer adjunct-therapy agent. The phytochemical research elucidated that the antitumor property of Ganoderma extract is due to its constituent triterpenes [terpenes . The stuterpenes . Zhang eterpenes .Natural compounds are proposed to compensate for the undesirable effects of chemotherapeutic drugs. Frequently, the bio-accessibility and bioavailability of natural agents, and their nontoxicity on normal cells and anti-tumor/anti-metastatic potentials nominates natural agents as propitious, multipotent chemopreventive and chemotherapeutic remedies. Various surveys on cells and animal models of TNBC revealed the suppressive efficacy of herbal medicine extracts and bioactive compounds including: flavonoids; terpenes; carotenoids; alkaloids; retinoids and lignans on various molecules of the Wnt/β-catenin signaling pathway, WNT, FZD, LRP, GSK3β, APC, Dsh, β-catenin and TCF/LEF. These phytotherapy agents exerted anti-proliferation, proapoptotic, anti-migration and anti-invasion effects and the inhibition of BCSC self-renewal via modulation of the Wnt downstream genes . The administration of a number of these herbal medications simultaneously repressed Wnt and mTORC1, ER stress and SATB1 signaling . The co-


Benefitting from the successful construction of bidirectional aligned three-dimensional conductive networks and electromagnetic synergistic effect, when the mass fraction of Ti3C2Tx and Fe3O4 are 2.96 and 1.48 wt%, BTFCA/epoxy nanocomposites show outstanding EMI shielding effectiveness of 79 dB, about 10 times of that of blended Ti3C2Tx@Fe3O4/epoxy (8 dB) nanocomposites with the same loadings of Ti3C2Tx and Fe3O4. Meantime, the corresponding BTFCA/epoxy nanocomposites also present excellent thermal stability (Theat-resistance index of 198.7 °C) and mechanical properties . Our fabricated BTFCA/epoxy nanocomposites would greatly expand the applications of MXene and epoxy resins in the fields of information security, aerospace and weapon manufacturing, etc. High‑efficiency electromagnetic interference (EMI) shielding materials are of great importance for electronic equipment reliability, information security and human health. In this work, bidirectional aligned TiThe online version contains supplementary material available at 10.1007/s40820-022-00949-8. With the rapid development of modern electronic information technology, especially for aerospace weapons and equipment technology, electromagnetic interference (EMI) pollution problem caused by high-frequency and high-power electronic equipment is becoming increasingly serious. It poses serious threats to normal operation of precise electronic components and human health –3. Polymσ) and easy processing, etc. Herein, Ti3C2Tx has been widely applied in the field of EMI shielding due to mature preparation technology and superior σ value [3C2Tx nanosheets tend to agglomerate inner polymer matrix, and have large contact resistance between the nanosheets, leading to higher percolating threshold of the composites [To our knowledge, polymer matrix EMI shielding composites have achieved satisfactory EMI shielding performances by adding highly conductive and/or magnetic fillers –9. The c σ value –23. Howemposites –26, whicmposites , 28.σ, EMI shielding effectiveness (EMI SE) and mechanical properties of polymer composites at relatively low Ti3C2Tx loadings [3C2Tx aerogel by freeze-drying method, and further impregnated epoxy resins to prepare Ti3C2Tx aerogel/epoxy composites. When the volume fraction of Ti3C2Tx was 0.40 vol%, the EMI SE of Ti3C2Tx aerogel/epoxy composites was 35 dB. Sun et al. [3C2Tx composites by electrostatic self-assembly and molding method. When the mass fraction of Ti3C2Tx was 4.0 wt%, σ and EMI SE of PS@Ti3C2Tx composites were 1081 S m−1 and 54 dB, respectively. In our previous work, Gu et al. [σ and EMI SE of the obtained CCA@rGO/PDMS composites reached 75 Sm−1 and 51 dB, respectively.Researches show that construction of three-dimensional (3D) conductive networks is proved to be an effective way to synchronously realize the excellent loadings –31. Shi loadings  preparedn et al.  preparedu et al.  obtainedσ [3C2Tx foams by directional freezing and further impregnated PDMS to prepare Ti3C2Tx foam/PDMS composites. When the mass fraction of Ti3C2Tx was 6.1 wt%, the σ and EMI SE of Ti3C2Tx foam/PDMS composites were 2211 S m−1 and 54 dB, respectively. Zhao et al. [3C2Tx/graphene hybrid aerogels (MGA) by directional freezing, and further impregnated epoxy resins to prepare MGA/epoxy composites. When the volume fraction of graphene and Ti3C2Tx was 0.18 and 0.74 vol%, the σ and EMI SE of MGA/epoxy composites were up to 695.9 S m−1 and 50 dB, respectively. Compared with the directional aligned 3D conductive networks, the bidirectional aligned 3D conductive networks can further reduce the percolating threshold and enhance the attenuation of electromagnetic waves by taking advantage of more regular internal interfaces [3C2Tx foams by bidirectional freezing method. The density of Ti3C2Tx foams was only 11.0 mg cm−3, EMI SE and SE/density (SSE) reached 71 dB and 8818 dB cm3 g−1 respectively, which exceeded the shielding performances of most reported foams. Sambyal et al. [3C2Tx/CNT foams by bidirectional freezing method. Results showed that the EMI SE of Ti3C2Tx/CNT foams (with density of only 2.5 mg cm−3) was up to 78 dB. Bidirectional aligned 3D Ti3C2Tx foam and Ti3C2Tx/CNT foam have been reported to exhibit excellent EMI shielding performances, but relatively poor mechanical properties have limited their broader applications. Cellulose nanofibers (CNF) possess excellent mechanical properties [3C2Tx via bidirectional freezing method by hydrogen bonds, which would be favor of enhancing the mechanical properties of Ti3C2Tx foams [Compared with the randomly dispersed 3D conductive networks, the aligned 3D conductive networks are not only conducive to further improving the σ –37, but σ –40. Wu eσ  preparedo et al.  preparedterfaces –44. Han terfaces  preparedl et al.  preparedoperties , 48, andTx foams –51.3O4 shows great application potential in EMI shielding materials due to excellent magnetism and low cost [In addition, for common polymer matrix EMI shielding composites, most electromagnetic waves are reflected at the interfaces between composites and air due to impedance mismatch, which would cause electromagnetic pollution to the service environment –54. Reselow cost –60.3C2Tx and Fe3O4 were firstly assembled by electrostatic interaction, followed by combined with CNF through hydrogen bonding, and bidirectional aligned Ti3C2Tx@Fe3O4/CNF aerogels (BTFCA) were then prepared by bidirectional freezing and freeze-drying technique. Finally, the BTFCA/epoxy nanocomposites were prepared by vacuum-assisted impregnation of epoxy resins. Structures and morphologies of BTFCA were characterized by X-ray diffraction (XRD), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Furthermore, the effects of Fe3O4 loadings on σ, EMI SE, thermal stabilities and mechanical properties of the BTFCA/epoxy nanocomposites were analyzed in detail, and the corresponding EMI shielding mechanism was investigated.In this work, Ti3O4 was positively modified by CTAB, followed by mixing with Ti3C2Tx dispersion and freeze-dried  for 36 h to get Ti3C2Tx@Fe3O4 hybrid. Different contents of Ti3C2Tx@Fe3O4 were dispersed in 10 mL of 2.5 mg mL−1 CNF in a glass vessel by a probe sonication for 10 min in an ice bath, followed by vigorous stirring for 3 h. Then the dispersion was poured in a square mold , and liquid nitrogen (− 196 °C) was used to freeze the bottom of the cylindrical mold through the intermediary of copper blocks. BTFCA was obtained by freeze-drying at −60 °C with pressure less than 5 Pa, followed by annealed at 400 °C for 2 h at a heating rate of 5 °C s−1 in an Ar + 5% H2 ambient. Ti3C2Tx content was fixed as 400 mg, the weight ratio of Ti3C2Tx/Fe3O4 was 4/1, 2/1, 1/1, and 1/2, respectively, and the corresponding samples were marked as BTFCA-1, BTFCA-2, BTFCA-3 and BTFCA-4. For comparison, the bidirectional Ti3C2Tx/CNF aerogels (BTCA) were also prepared.FeEpon 862 and diethyl methyl benzene diamine were firstly stirred at 70 °C for 1 h, and then filled into BTFCA via vacuum-assisted impregnation technique. Finally, BTFCA/epoxy nanocomposites were prepared by heating at 120 °C for 5 h. For comparison, BTCA/epoxy nanocomposites were prepared by the same process. Figure 3AlC2 precursor with dense layered structure  is exfoliated into few-layered Ti3C2Tx nanosheets . In Fig. S2, Fe3O4 presents (220), (311), (400), (422), (511), and (440) diffraction peaks [−1. After electrostatic assembly, Fe3O4 is uniformly dispersed on Ti3C2Tx nanosheets to obtain Ti3C2Tx@Fe3O4 . Figure 3C2Tx and (002) lattice plane of CNF [3O4 is introduced, three new diffraction peaks appear at 36.7°, 43.6°, and 63.8° of BTFCA-2, corresponding to the (311), (400) and (440) crystal planes of Fe3O4, respectively. As observed in Fig. −1, which are attributed to the graphitized structures [3C2Tx appear within 100 ~ 700 cm−1. Peak at 198 cm−1 is attributed to A1g symmetry out-of-plane vibration of Ti atom. Peaks at 374 and 583 cm−1 are ascribed to the Eg group vibration and in-plane (shear) modes of Ti, C, and surfapresentsce functional groups [3O4 is introduced, a new peak appears in BTFCA-2 at 655 cm−1, which is attributed to the vibration of Fe3O4 in A1g mode. As shown in Fig. p, Ti 3s, C 1s, Ti 2p, O 1s, Ti 2s, and F 1s at 35, 60, 287, 457, 531, 563, and 685 eV [3O4 is introduced, BTFCA-2 shows two new peaks at 709 and 723 eV, which are attributed to Fe 2p3/2 and Fe 2p1/2, respectively. From Fig. −1. XRD, Raman spectroscopy, XPS and hysteresis loops indicate that BTFCA has been successfully prepared.In Fig. S1, Tion peaks , and core of CNF . After Fructures  formed al groups . After Fd 685 eV . After F3C2Tx and CNF in BTCA support each other, to form the long-range aligned lamellar structures. After Fe3O4 is introduced, BTFCA still maintains the long-range aligned lamellar structures. With low loadings of Fe3O4, the cell size of BTFCA increases slightly  nanocomposites, and also significantly higher than that of blended Ti3C2Tx@Fe3O4/epoxy  nanocomposites with the same loadings of Ti3C2Tx and Fe3O4. Highly conductive Ti3C2Tx nanosheets are aligned along the axial and radial directions in BTCA and BTFCA to construct a bidirectional aligned 3D conductive network, which enhances the contact among Ti3C2Tx nanosheets and forms abundant conductive paths, thus showing outstanding σ. The introduction of Fe3O4 would affect the contact among Ti3C2Tx nanosheets and hinder formation of Ti3C2Tx-Ti3C2Tx conductive paths, leading to slightly reduced σ of BTFCA/epoxy nanocomposites. The addition of excessive Fe3O4 reduces the overlap among Ti3C2Tx nanosheets and significantly increases the cell size of BTFCA, which severely restricts the formation of bidirectional aligned 3D conductive networks for BTFCA, resulting in significant decrease of σ.Figure 3O4. When the mass fraction of Fe3O4 is 1.48 wt%, EMI SE of BTFCA-2/epoxy nanocomposites is 79 dB, 11.3% higher than that of BTCA/epoxy (71 dB) nanocomposites, and also about 10 times that of blended Ti3C2Tx@Fe3O4/epoxy  nanocomposites with the same loadings of Ti3C2Tx and Fe3O4. The bidirectional aligned 3D conductive networks of BTFCA/epoxy nanocomposites provide abundant moving loads such as charge carriers. Under alternating electric field, they can induce microcurrent to form electrical loss through tunneling effect and other ways, and convert the energy of electromagnetic waves into heat. At the same time, the internal complex heterogeneous interfaces in BTFCA/epoxy nanocomposites extend the transmission paths of electromagnetic waves, which are conducive to enhancing the scattering and reabsorption of electromagnetic waves and further dissipating electromagnetic waves. After introduction of Fe3O4, reduced overlaps among Ti3C2Tx nanosheets lead to gradually decreased σ of BTFCA/epoxy nanocomposites and weakened electrical loss (such as ohmic loss) is not conducive to improvement of EMI SE. On the other hand, BTFCA/epoxy nanocomposites construct 3D magnetic networks, which enhances the multiple reflection and reabsorption of electromagnetic waves and strengthens the magnetic hysteresis loss and other magnetic losses of electromagnetic waves, so as to improve the dissipation ability of electromagnetic waves. In addition, the introduction of Fe3O4 brings more heterogeneous interfaces. Due to interface polarization, there are a large number of dipoles at heterogeneous interfaces, which will cause polarization loss to electromagnetic waves and further enhance the attenuation of electromagnetic waves. As a result, BTFCA-2/epoxy nanocomposites present the best EMI shielding performances. And the corresponding schematic illustration of EMI shielding mechanism of BTFCA/epoxy nanocomposites is shown in Fig. Figure SET, SEA and SER of BTCA/epoxy and BTFCA/epoxy nanocomposites. With increasing loadings of Fe3O4, SER of BTFCA/epoxy nanocomposites decreases gradually, and SEA increases first and then decreases. When the mass fraction of Fe3O4 is 1.48 wt%, SER and SEA of BTFCA-2/epoxy nanocomposites are 8 and 71 dB respectively. With increasing loadings of Fe3O4, the gradually decreased σ of BTFCA/epoxy nanocomposites improves the impedance matching, resulting in gradual decrease of SER. Although the electrical loss of BTFCA/epoxy nanocomposites to electromagnetic waves is gradually weakened, the internal multiple reflection, magnetic loss and polarization loss are gradually enhanced. As a result, SEA of BTFCA-2/epoxy nanocomposites is the maximum.Figure 3O4, the electromagnetic wave effective absorbance of BTFCA/epoxy nanocomposites gradually increases. When the mass fraction of Fe3O4 is 1.48 wt%, the electromagnetic wave effective absorbance of BTFCA-2/epoxy nanocomposites is 89.5%. The introduction of Fe3O4 not only improves the impedance matching of BTFCA/epoxy nanocomposites and air, but also enhances the magnetic loss to improve the electromagnetic wave effective absorbance.Figure R), absorption (A) and transmission (T) coefficients of BTCA/epoxy and BTFCA/epoxy nanocomposites. With increasing loadings of Fe3O4, the R coefficient of BTFCA/epoxy nanocomposites decreases gradually, and the T coefficient decreases first and then increases. When the mass fraction of Fe3O4 is 1.48 wt%, the T coefficient of BTFCA-2/epoxy nanocomposites is the lowest, only 4 × 10–4, and the R coefficient is 0.78. It demonstrates that the incorporation of Fe3O4 can significantly improve the EMI shielding performances. With increasing loadings of Fe3O4, the σ of BTFCA/epoxy nanocomposites decreases gradually, which improves the impedance matching between BTFCA/epoxy nanocomposites and the air, and reduces the R coefficient gradually, thus weakening the secondary electromagnetic pollution. With introduction of Fe3O4, although the decreased σ leads to reduced electrical loss to electromagnetic waves, the internal multiple reflection, magnetic loss and interfacial polarization loss are enhanced. Under the comprehensive action, BTFCA-2/epoxy nanocomposites show the optimal EMI shielding performances and the corresponding T coefficient is the lowest.Figure Figure Theat-resistance index (THRI) can reflect the heat resistance of the materials [THRI of BTFCA/epoxy nanocomposites increases gradually with increasing loadings of Fe3O4. When the mass fraction of Fe3O4 is 1.48 wt%, THRI of BTFCA-2/epoxy nanocomposites is 198.7 °C, increased by 2.6 °C compared with that of BTCA/epoxy (196.1 °C) nanocomposites. The main reason is that Fe3O4 has more excellent heat resistance and relatively lower weight loss, resulting in slightly improved thermal stabilities of BTFCA/epoxy nanocomposites.Figure aterials . It can δ of BTCA/epoxy and BTFCA/epoxy nanocomposites, the corresponding data are given in Tab. S2. The storage modulus of BTFCA/epoxy nanocomposites increases first and then decrease with increasing loadings of Fe3O4. When the mass fraction of Fe3O4 is 1.48 wt%, the storage modulus of BTFCA-2/epoxy nanocomposites is 9902.1 MPa, higher than that of BTCA/epoxy (9137.3 MPa) nanocomposites. It is due to that the introduction of Fe3O4 increases the surface roughness of BTFCA, which is conducive to enhancing interfacial strength between BTFCA and epoxy resins, thus improving the storage modulus. However, the addition of excessive Fe3O4 would destroy the bidirectional aligned 3D networks of BTFCA, leading to decreased storage modulus.Figures 3O4. When the mass fraction of Fe3O4 is 1.48 wt%, the Young's modulus and hardness of BTFCA-2/epoxy nanocomposites are 4.51 and 0.34 GPa, improved by 6.9% and 6.3% than those of BTCA/epoxy (4.23 and 0.32 GPa) nanocomposites. The reason is that the high stiffness and hardness of Fe3O4 can improve the Young's modulus and hardness of BTFCA/epoxy nanocomposites. However, the addition of excessive Fe3O4 would damage the bidirectional aligned 3D networks of BTFCA and weaken the ability of BTFCA/epoxy nanocomposites to resist deformation under external force, resulting in decreased Young’s modulus and hardness.Figure 3C2Tx and Fe3O4 is 2.96 and 1.48 wt%, BTFCA/epoxy nanocomposites show outstanding EMI SE of 79 dB, about 10 times of that of blended Ti3C2Tx@Fe3O4/epoxy (8 dB) nanocomposites with the same loadings of Ti3C2Tx and Fe3O4. Moreover, BTFCA/epoxy nanocomposites also present excellent thermal stability (THRI of 198.7 °C) and mechanical properties . The obtained BTFCA/epoxy nanocomposites would greatly expand the applications of MXene and epoxy resins in the fields of information security, aerospace and weapon manufacturing, etc.Bidirectional aligned BTFCA and the corresponding BTFCA/epoxy nanocomposites with long-range aligned lamellar structures were successfully prepared. Benefitting from the successful construction of bidirectional aligned 3D conductive networks and electromagnetic synergistic effect, when the mass fraction of TiSupplementary file1 (PDF 613 KB)Below is the link to the electronic supplementary material.


Computational and Mathematical Methods in Medicine has retracted the article titled “Effects of Afatinib on Development of Non-Small-Cell Lung Cancer by Regulating Activity of Wnt/β-Catenin Signaling Pathway” [Pathway”  due to cFollowing an investigation conducted by the Hindawi Research Integrity team , signifi


Knowledge of the role of CYP2E1 in hepatocarcinogenesis is largely based on epidemiological and animal studies, with a primary focus on the role of CYP2E1 in metabolic activation of procarcinogens. Few studies have directly assessed the effects of CYP2E1 on HCC malignant phenotypes.The expression of CYP2E1 in HCC tissues was determined by qRT-PCR, western blotting and immunohistochemistry. Overexpression of CYP2E1 in HCC cell was achieved by lentivirus transfection. The function of CYP2E1 were detected by CCK-8, wound healing, transwell assays, xenograft models and pulmonary metastasis model. TOP/FOPFlash reporter assay, western blotting, functional rescue experiments, Co-immunoprecipitation and reactive oxygen species detection were conducted to reveal the underlying mechanism of the tumor suppressive role of CYP2E1.CYP2E1 expression is down-regulated in HCC tissues, and this downregulation was associated with large tumor diameter, vascular invasion, poor differentiation, and shortened patient survival time. Ectopic expression of CYP2E1 inhibits the proliferation, invasion and migration and epithelial-to-mesenchymal transition of HCC cells in vitro, and inhibits tumor formation and lung metastasis in nude mice. Mechanistic investigations show that CYP2E1 overexpression significantly inhibited Wnt/β-catenin signaling activity and decreased Dvl2 expression in HCC cells. An increase in Dvl2 expression restored the malignant phenotype of HCC cells. Notably, CYP2E1 promoted the ubiquitin-mediated degradation of Dvl2 by strengthening the interaction between Dvl2 and the E3 ubiquitin ligase KLHL12 in CYP2E1-stable HCC cells. CYP2E1-induced ROS accumulation was a critical upstream event in the Wnt/β-Catenin pathway in CYP2E1-overexpressing HCC cells.These results provide novel insight into the role of CYP2E1 in HCC and the tumor suppressor role of CYP2E1 can be attributed to its ability to manipulate Wnt/Dvl2/β-catenin pathway via inducing ROS accumulation, which provides a potential target for the prevention and treatment of HCC.The online version contains supplementary material available at 10.1186/s12967-022-03396-6. Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death globally and its incidence continues to rise . CurrentCytochrome P450 (CYP) enzymes are a superfamily of hemoproteins and extensively catalyze the metabolism of exogenous and endogenous compounds. CYP also exerts a crucial role in the development and progression of cancer, diabetes, heart disease, and Parkinson's disease –5. CYP2EHowever, the most common etiology of HCC is viral infection (hepatitis B virus and hepatitis C virus), not procarcinogens. Especially in China, 85% of HCC cases are associated with chronic HBV infection , 10. OurCTNNB1 (β-catenin) mutations are elevated with HCC progression . At 35 days after injection, the nude mice were euthanized and tumors were collected for the subsequent orthotopic liver tumor model. These animal experiments were approved by the Animal Ethics Committee of Zhengzhou University and carried out in accordance with the guidelines of animal ethics.Four-week-old male BALB/c nude mice were provided by the Beijing Vital River Laboratory Animal Technology Company. After habituation for 3 days, 100 μl of MHCC-97H-GFP or MHCC-97H-CYP2E1 cell suspension . The left lobe of the liver of the 4.5-week-old male mice was extruded under anesthetization and a tunnel was made by a 10 ml syringe needle. Then 2 pieces of the tumors were deposited into the left lobe through the tunnel. Subsequently, the left lobe of the liver was returned to the abdominal cavity, and the muscle and skin were sutured. Ten weeks after the operation, the livers were removed and orthotopic tumors were counted.6 cells/ml) were injected into the tail vein of 4.5-week-old mice. At the 9th week the nude mice were euthanized and the lungs removed. The lungs were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into slices. The slices were moved onto glass slides, dewaxed, rehydrated in ethanol solution and followed by the addition of hematoxylin and eosin to determine the metastasis of cancer cells.200 μl of MHCC-97H-GFP or MHCC-97H-CYP2E1 cell suspension  < 0.05 indicated statistically significant.SPSS version 21.0 and GraphPad Prism 5.0 were used to perform statistical analysis and generate graphs, respectively. The Kolmogorov–Smirnov and Shapiro–Wilk methods were used to assess the normality of the data. For normal distribution data, a two-tailed Student’s P = 0.049)   -dependent transcriptional activity both in MHCC-97H and SMMC-7721 cells Fig. A. Next, Dvl2 plays a core role in the Wnt signaling pathway . The aboA unique property of CYP2E1 is to produce reactive oxygen species (ROS). We investigated whether CYP2E1-mediated inhibition of the HCC phenotype as well as Wnt/β-catenin pathway is ROS-dependent. An increased level of intracellular ROS was seen in both MHCC-97H-CYP2E1 and SMMC-7721-CYP2E1 cells in comparison with their corresponding control cells Fig. A. TreatmInterestingly, CYP2E1-overexpressing cells show an epithelial cobblestone-like morphology Fig. A. ConsidOur knowledge of the role of CYP2E1 in hepatocarcinogenesis is based on epidemiological and animal studies, with a primary focus on the role of CYP2E1 in the metabolic activation of procarcinogens; few studies have directly assessed the effects of CYP2E1 on HCC malignant phenotypes. Ho et al.  reportedc-Myc, Axin2, LEF1. Dvl is therefore the central component of Wnt/β-catenin signaling and relays signals from Wnt receptors to β-catenin by rescuing β‐catenin from degradation [Dvl1, Dvl2 and Dvl3), which exhibit a high degree of similarity, have been found in humans [Wnt/β-catenin signaling is an evolutionarily highly conserved pathway and plays decisive roles in development and tissue homeostasis. Aberrant activation of the Wnt/β-catenin signaling cascade is one of the most frequent molecular events in HCC  and condradation , 29. Thrn humans . Increasn humans –36. A ren humans .In the present study, CYP2E1 overexpression significantly inhibited Wnt/β-catenin signaling activity in HCC cells and Dvl2 expression level was reduced in CYP2E1-overexpressing HCC cells. Restoration of Dvl2 expression rescued the malignant phenotype of HCC cells. Previous studies have verified that Dvl2 is mainly regulated at the protein level by the ubiquitin–proteasome pathway. We thus speculated that the CYP2E1-blocked Wnt/β-catenin signaling pathway might depend on Dvl2 stability. KLHL12–Cullin-3 ubiquitin ligase was reported to mediate ubiquitination and degradation of Dvl2 under different physiological conditions , 38. Our2O2 negatively regulates the Wnt pathway by downregulating β-catenin [How does CYP2E1 regulate Wnt/β-Catenin signaling? CYP2E1 is one of the most active ROS-generating CYP isoforms and can produce high levels of ROS . ROS are-catenin . Combret-catenin .A recent finding revealed that oxidative stress suppressed distant metastasis by melanoma cells in vivo . Large-sCollectively, our studies provide novel insight into the role of CYP2E1 in HCC, independent of its activation of procarcinogens and mutagens. Ectopic expression of CYP2E1 inhibits cell proliferation, migration, invasion and EMT of HCC cells in vitro and inhibited tumor formation and lung metastasis in nude mice. The protective effect of CYP2E1 is attributed to its ability to manipulate Wnt/Dvl2/β-catenin signaling through ROS accumulation, which provides a potential target for the prevention and treatment of HCC.Additional file 1: Table S1. Donor characteristics of human liver samples. Table S2. Primers for quantitative real-time polymerase chain reaction.Additional file 2: Fig. S1. The expression level of CYP2E1 mRNA in HCC cells.Additional file 3: Fig. S2. Statistical graphs of ROS level in control and CYP2E1-overexpressing HCC cells.


Denosumab is a game-changing drug for giant cell tumor of bone (GCTB); however, its clinical biomarker regarding tumor ossification of GCTB has not been elucidated. In this study, we investigated the relationship between Wnt/β-catenin signaling and the ossification of GCTB and evaluated whether endogenous nuclear β-catenin expression predicted denosumab-induced bone formation in GCTB. Genuine patient-derived primary GCTB tumor stromal cells exhibited osteoblastic characteristics. Identified osteoblastic markers and nuclear β-catenin translocation were significantly upregulated via differentiation induction and were inhibited by treating with Wnt signaling inhibitor, GGTI-286, or selective Rac1-LEF inhibitor, NSC23766. Furthermore, we reviewed the endogenous ossification and nuclear β-catenin translocation of 86 GCTB clinical samples and elucidated that intra-tumoral ossification was significantly associated with the nuclear translocation. Three-dimensional quantitative analyses (n = 13) of tumoral CT images have revealed that the nuclear β-catenin translocation of naïve GCTB samples was significantly involved with the denosumab-induced tumor ossification. Our findings suggest a close relationship between the nuclear β-catenin translocation and the osteoblastic differentiation of GCTB. Investigations of the nuclear β-catenin in naïve GCTB samples may provide a promising biomarker for predicting the ossification of GCTB following denosumab treatment. The chief components of GCTB are osteoclast-like giant cells (GCTB-OCs) that express RANK and fibroblast-like spindle stromal cells that express RANKL, a key mediator of osteoclast activation 8. Recently, Histone 3.3 G34W (H3G34W), a driver mutation in H3 histone family member 3A (encoded by H3F3A), was identified as a specific surrogate marker of GCTB 10. The presence of this mutation confirmed that GCTB stromal cells (GCTB-SCs) are the actual neoplastic component of this tumor 11.Giant cell tumor of bone (GCTB) is a bone neoplasm characterized by locally aggressive and massive bone destruction 14. However, curative resection for GCTB in the axial skeleton  is not often feasible 14. Denosumab, a fully human monoclonal antibody that inhibits RANKL, was recently approved for the treatment of GCTB 18. Denosumab inhibits RANKL, thereby preventing RANK–RANKL interactions, resulting in deletion of GCTB-OCs and decreasing tumor-induced osteolysis 18. In addition, diffuse bone formation with the peripheral sclerotic rim is often observed after denosumab treatment 4. These histological and clinical consequences can result in a more comfortable operative procedure and decrease surgical morbidity 17. However, the underlying mechanism of that ossification was not known.Surgical resection has been the treatment of choice for GCTB 20. Consistent with this observation, several previous studies reported that GCTB-SCs have the ability to undergo osteoblastic differentiation 22. One essential requirement for osteoblastic differentiation could be the association between WNT/β-catenin signaling and its target gene, Runt-related transcription factor2 (RUNX2) 25. Previous studies reported nuclear β-catenin translocation and expression of RUNX2 in GCTB-SCs 26. These results suggest the involvement of the so-called "canonical" Wnt signaling pathway in the osteoblastic differentiation of GCTB-SCs. However, this hypothesis has remained unproven 7, and no useful markers have been identified to predict bone formation in GCTB.Although osteolysis is the characteristic feature of GCTB, intra-tumoral and peripheral bone develops in 30–50% of cases of GCTB In this study, we investigated the relationship between the Wnt/β-catenin signaling, a critical pathway for osteogenic differentiation, and ossification of GCTB using patient-derived primary cultures. In addition, we investigated whether nuclear β-catenin translocation in GCTB-SCs predicted bone formation after denosumab treatment.27.In patients with GCTB, administration of denosumab often induced massive bone formation, as shown in Fig. ALP, COL1A1, IBSP , RUNX2, and BGLAP  were significantly increased by the presence of OGM . As shown in Fig. OGM Fig. . OsteoblOGM Fig. ,S2d. Flu30. In addition, previous studies reported nuclear β-catenin translocation in GCTB-SCs 31. Hence, we hypothesized that the canonical Wnt pathway regulates the osteoblastic differentiation of pGCTB-SCs. Accordingly, we grew pGCTB-SCs in OGM, lysed the cells, and purified cytoplasmic and nuclear protein fractions. By western blotting, we confirmed that the nuclear β-catenin translocation was upregulated within 12 h after OGM treatment, although cytoplasmic β-catenin remained unchanged , a recently identified Wnt/β-catenin pathway inhibitor 33. Furthermore, Rac1 promotes the nuclear β-catenin translocation through phosphorylation at Ser191 by a downstream effector kinase, JNK 34. Hence, we analyzed the effect of a selective Rac1-GEF inhibitor, NSC23766 (NSC), on OGM-induced osteoblastic differentiation of pGCTB-SCs. Like GGTI, NSC also decreased the expression levels of ALP and other osteoblastic markers in pGCTB-SCs following OGM treatment , a method to evaluate nuclear β-catenin 47, in GCTB-SCs using 91 clinical samples of GCTB. Of those samples, three cases were excluded due to overlap, one was removed due to low sample quantity, and another was excluded because the associated patient data were not available. Ultimately, a total of 86 tumor sections were retrospectively reviewed. The background data on these sections are presented in Supplementary Table 2 = 0.877 by Pearson product-moment correlation coefficient; P < 0.001, Fig. 20. Remarkably, the positive group exhibited a significantly higher rate of intra-tumoral ossification .Some cases of GCTB exhibit spontaneous intra-tumoral ossification, whereas others do not The diversity of endogenous nuclear β-catenin translocation in GCTB-SCs may explain differences in bone formation after denosumab treatment. To address this issue, we studied 14 consecutive cases of GCTB that received denosumab. One case was excluded due to a problem with the specimen; the remaining 13 tumor samples were reviewed. Detailed information on individual cases is summarised in Table First, we examined NBLI in GCTB-SC using naïve GCTB samples prior to denosumab administration. Seven cases were positive and six were negative. Case #5, a positive group representative, exhibited massive bone formation after denosumab treatment, as revealed in CT images Fig. a and b. 36. In the window setting where the tumor margins were discernible, ROIs for histogram analyses were semi-automatically delineated by tracing the tumors' outer margins before , indicating that GCTB-SCs did not retain pluripotency. During osteoblastic differentiation, the canonical Wnt/β-catenin signaling is the dominant pathway 32. In addition, Rac1 stimulates nuclear β-catenin translocation through phosphorylation at Ser191 by a downstream effector kinase, JNK 27. Consistent with these previous results, we found that Rac1 inhibition suppressed nuclear β-catenin translocation and osteoblastic differentiation of GCTB-SCs. Therefore, we considered that denosumab administration caused activation of Rac1 and triggered the nuclear localization of β-catenin, followed by osteoblastic differentiation of GCTB-SCs, ultimately resulting in the cessation of tumor activity. If this is the case, forced activation of Rac1 in GCTB-SCs by a potent Rac1-agonist, such as the recently discovered natural polyketide deacetylmycoepoxydiene 38, may stimulate the osteoblastic differentiation of GCTB-SCs, and combination treatment with denosumab and Rac1-agonist could be an effective strategy for the treatment of GCTB.GGTI inhibits protein prenylation, and this process is essential for the correct localization and functions of GTPases, including Rac1 39. However, extrinsic inhibition of GSK3β by LiCl did not play significant roles in osteoblastic differentiation of GCTB-SCs, as shown in Fig. We detected baseline nuclear β-catenin translocation in pGCTB-SCs, indicating intrinsic activation of canonical Wnt/β-catenin signaling. Interestingly, a previous study showed that miR-125a stimulates the translocation of β-catenin in GCTB-SCs through GSK3β-mediated canonical signaling, resulting in cell proliferation and tumorigenicity 40. Expression of the Wnt inhibitor secreted frizzled-related protein (sFRP) in GCTB-OCs has been confirmed by comprehensive mRNA profiling of GCTB-SCs 41. More importantly, we observed expression of Wnt-5a in GCTB-SCs (data not shown). Based on these findings, we hypothesized that deletion of GCTB-OCs by denosumab might decrease the level of sFRP in tumor tissues, activate a cascade of Wnt-5a/Ror2/Rac1 signaling, and finally cause the osteoblastic differentiation of GCTB-SCs.Several stimuli can elicit Rac1 activation. For example, both Wnt-5a (a Wnt family member) and Ror2  activate Rac1 and induce differentiation of human mesenchymal stem cells into osteoblasts 42. Therefore, we speculated that GCTB-SCs with nuclear β-catenin translocation would correspond to S1 cells and be partly committed to differentiation into osteoblasts. Therefore, as discussed in a previous paragraph, the deletion of GCTB-OCs by denosumab triggered the cells' final osteoblastic differentiation. However, this notion should be further investigated.As another essential feature of this study, we found that endogenous nuclear β-catenin translocation was associated with osteoblastic differentiation of GCTB-SCs via denosumab treatment. Besides, upregulated NBLI was correlated with endogenous intra-tumoral bone formation in GCTB. Notably, a recent epigenetic analysis showed that GCTB-SCs could be classified into three groups, S1 to S3. S1 cells are characterized by the expression of osteoblast-associated genes such as osteopontin, whereas S3 cells have markers of the myofibroblastic lineage, e.g., alpha-smooth muscle actin. S2 cells have features intermediate between those of S1 and S3 cells This study had several limitations. First, the precise mechanism by which Rac1 activation occurs in GCT-SCs following denosumab treatment was not fully elucidated. More detailed in vitro experiments, including co-cultures of GCTB-SCs and GCTB-OCs, are needed to clarify this issue. Secondly, the number of patients who received denosumab treatment was relatively small in a retrospective analysis. This is because the rarity of GCTB and the recent approval of denosumab for GCTB treatment. To further validate the ability of the NBLI in GCTB-SCs to predict bone formation after denosumab treatment, we plan to conduct more extensive prospective studies in the future.In summary, our findings suggest a close relationship between the nuclear β-catenin translocation via Rac1 activation and the osteoblastic differentiation of GCTB. Investigations of NBLI in naïve GCTB samples will provide a promising biomarker for predicting the degree of bone formation after denosumab treatment.Monoclonal rabbit anti-H3G34W antibody (clone RM263) was purchased from RevMab Biosciences . Monoclonal rabbit anti-β-catenin antibody (#32572), anti-alkaline phosphatase, tissue non-specific antibody (#126820), goat anti-rabbit IgG H&L preabsorbed , and goat anti-mouse IgG H&L preabsorbed  were purchased from Abcam . Monoclonal mouse anti-actin clone C4 antibody (MAB1501) was purchased from Merck Millipore . Monoclonal mouse anti-RUNX2 antibody (sc-390351) was purhchased from Santa Cruz Biotechnology . A selective inhibitor of geranylgeranyltransferase1, GGTI-286 (#22756), was purchased from Cayman Chemical , and a selective Rac1  inhibitor, NSC23766, was purchased from Merck Millipore.43. Briefly, antigen retrieval of deparaffinized, rehydrated sections was performed by heating in a microwave for 20 min with 10 mM citric acid at pH 6.0 (FUJIFILM Wako). After blocking with normal goat serum for 30 min at room temperature, the sections were incubated with anti-H3G34W monoclonal Abs (1:200) at 4 °C overnight 44. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxidase in methanol for 30 min at room temperature, and specimens were then incubated with Dako EnVision™+ Dual Link System-HRP  for 45 min at room temperature. Antibody complex was visualized using the diaminobenzidine substrate system (FUJIFILM Wako) and counterstained with hematoxylin 44. Section images were obtained on a Keyence BZ-X800 microscope , and nuclear immunoreactivity for H3G34W was evaluated.Surgically-resected GCTB clinical samples were fixed with 4% PFA and embedded in paraffin (FFPE). Immunostaining was performed as described previously FFPE sections (not exposed to denosumab) were deparaffinized, rehydrated, and incubated with adjusted solutions for 15 min at 37℃ according to the manufacturer's protocol  for the detection of TRAP activity of GTCB-OCs. The stained sections were washed three times with deionized water and counterstained with Methyl Green Solution (FUJIFILM Wako) for 5 s. Denosumab-exposed tissue samples were also stained as negative controls.Adjacent pre-exposed FFPE sections were immunohistochemically stained for H3G34W and RUNX2, the master regulator of osteogenesis, to evaluate the osteogenic potential of GCTB-SCs, using ImmPRESS® Duet Double Staining Polymer Kit . Briefly, antigen unmasking of deparaffinized, rehydrated sections was performed with citrate-based solutions at pH 6.0, and endogenous peroxidase activity was blocked by incubation with BLOXALL® Blocking Solution (Vector Laboratories) for 20 min, followed by incubation with 2.5% normal horse serum for 20 min at room temperature. The adjacent specimens were incubated with anti-H3G34W rabbit monoclonal antibody (1:200) or anti-RUNX2 mouse monoclonal antibody (1:200) at 4 °C overnight and then incubated with ImmPRESS Duet Reagent (Vector Laboratories) for 10 min at room temperature. Antibody complex was visualized by incubation with the ImmPACT DAB EqV Substrate (Vector Laboratories) for 2 min against H3G34W mutation or the ImmPACT Vector Red Substrate (Vector Laboratories) for 20 min against RUNX2.2 and 95% air. GCTB-OCs were present only in the first passage, whereas primary GCTB-SCs (pGCTB-SCs) were further amplified. Upon reaching confluence, pGCTB-SCs were sub-cultured, and the third through eighth passages were used for subsequent experiments. To induce osteogenic differentiation, confluent cells were treated with osteogenic medium (OGM) containing Minimum Essential Medium (Thermo Fisher Scientific) supplemented with 10% FBS, 100 nM dexamethasone , 100 μM ascorbic acid (FUJIFILM Wako), and 10 mM β-glycerophosphoric acid  for various periods.Some fresh GCTB tumor samples were obtained from surgeries and washed with warmed PBS. The samples were minced in DMEM  supplemented with 10% FBS , 100 U/ml penicillin, and 100 μg/ml streptomycin. Together with small pieces of chipped tissues, the cell suspension was transferred to culture dishes and cultured at 37 °C in a humidified atmosphere of 5% CO2 (FUJIFILM Wako), and the blocks were embedded in paraffin and immunohistochemically stained for H3G34W mutation.To determine the purity of primary cultures, we prepared cell blocks and performed immunostaining with anti-H3G34W. Confluent suspensions of pGCTB-SCs were harvested, and cell pellets were prepared by centrifugation for 5 min at 1,500 rpm. The pellets were incubated overnight at 37 °C in DMEM, and then fixed for 3 h at room temperature (RT) in 10% Formalin Neutral Buffer Solution (FUJIFILM Wako). After fixation, the supernatant was aspirated, and 1% sodium alginate (FUJIFILM Wako) was added to the pellets. Gelatinous cell blocks were immediately obtained by addition of 100 μl of 1 M CaCl4 cells per well. After 48 h of incubation, the culture medium was exchanged and further incubated for the indicated periods. The cells were washed with PBS, fixed in 10% formalin, and stained with premixed ALP substrate solutions (FUJIFILM Wako).ALP activity is widely used to assess the early osteogenic ability of osteoblast-like cells. We seeded pGCTB-SCs into a 24-well plate at a density of 5 × 103 cells per well and incubated for 48 h. After the cells reached confluence, the medium was exchanged, and the samples were incubated further. ALP assays were performed using the TRACP & ALP assay kit . Briefly, treated cells were lysed in extraction solution , mixed with freshly prepared p-nitrophenyl phosphate substrate (12.5 mM), and incubated at 37 °C for 30 min. The optical density of p-nitrophenol at 405 nm was determined using iMark™ Microplate Absorbance Reader . Finally, ALP activity was normalized with a standard curve derived from Escherichia coli C75 (Takara Bio).pGCTB-SCs were seeded into 96-well plates at a density of 5 × 10GAPDH. At least four separate experiments were conducted.Total RNA of treated pGCTB-SCs was extracted using the RNeasy Mini Kit  and reverse-transcribed with PrimeScript RT Reagent Kit (Takara Bio). qRT-PCR was conducted with a LightCycler 1.5  with the TB Green® Premix Ex Taq™ II (Takara Bio). Primers are listed in Supplementary Table 4 cells and cultured for 48 h. After the cells were treated with each reagent for the indicated periods, they were fixed with 4% paraformaldehyde (FUJIFILM Wako) for 10 min at RT, permeabilized with 0.2% Triton X-100  for 15 min, and blocked with 10% goat serum (FUJIFILM Wako) for 30 min. Subsequently, the cells were incubated at 4 °C overnight with a mixture of primary antibodies diluted in 1:200 in Can Get Signal Immunostain Solution A . Samples were then washed three times with PBS and incubated with Alexa Fluor® 488 and 594 diluted in 1:200 for 1 h at RT. SlowFade Diamond antifade mountant with DAPI (Invitrogen) was used as a mounting solution. Immunostaining was visualized using fluorescence microscopy .pGCTB-SCs were seeded on poly-L-lysine (Fujifilm Wako)–coated cover glass at a density of 10 × 106 cells/well density and incubated overnight. The following day, the culture media were replaced for each reagent, and the cells were incubated for an additional 12 h. After incubation, the cells were washed twice with ice-cold PBS, scraped, and centrifuged. Cytoplasmic and nuclear proteins were isolated using nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific), to which Cell Lytic M (Sigma-Aldrich) with protease inhibitor cocktail (cOMplete™ Mini: Sigma-Aldrich) were added.pGCTB-SCs were seeded in 6-well dishes at a 1.2 × 1045 with the following primary antibodies: β-catenin (1:1000) and actin clone C4 (1:5000), with or without rabbit polyclonal Lamin A/C antibody . Relative intensity was calculated using the ratio of each target protein's signal intensity to internal controls' intensity, using ImageJ ver1.52p . At least three separate experiments were conducted. Full-length blots were absent because membranes were cut prior to hybridization with primary antibodies. The unedited blots including replicates were shown in Supplementary Figs. Western blotting was performed as previously described All the methods were conducted in accordance with the Declaration of Helsinki and written informed consent was obtained from all human subjects.44. A total of 91 clinical samples of GCTB from 88 patients were prepared for immunohistochemistry. These tumor specimens had been acquired from biopsy or surgeries, and the existence of the H3G34W mutation had been immunohistochemically confirmed. Samples collected after denosumab treatment were excluded from the study.To evaluate nuclear β-catenin translocation in naïve GCTB clinical samples, we performed a retrospective analysis using samples of GCTB registered in the files of the Department of Anatomic and Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan 47. Histogram analysis was conducted to calculate the cut-off value for the NBLI. The presence of intra-tumoral ossification of GCTB was also assessed using H&E-stained sections.Immunohistochemical staining and assessment of the nuclear β-catenin labeling index (NBLI) were performed as previously described 48. In addition, we assessed the association between nuclear β-catenin translocation and intra-tumoral ossification of GCTB.Twenty-one patients were diagnosed with GCTB or received treatment for this cancer at our hospital between July 2011 and November 2020. Of those, 18 patients had received denosumab treatment , and 16 had also undergone non-contrast CT or PET-CT evaluation before and after denosumab treatment. CT DICOM  image datasets from identified patients were analyzed using SYNAPSE VINCENT ver6.1 . A single musculoskeletal radiologist with eight years of experience manually delineated the regions of interest (ROI) in the axial CT images of whole slices, and three-dimensional CT images were semi-automatically acquired. Slice thickness was set at 2 or 5 mm. We identified the calcified tissue volumes  using VINCENT histogram analysis and quantified tumor calcification using the previously described Agatston scoring system P < 0.05 was considered statistically significant.All experiments were repeated at least three times. Data are presented as means ± SD. Student’s t-test or Wilcoxon's rank-sum test was used for two-group comparisons. Using one-way ANOVA with the Tukey–Kramer post hoc test, multiple comparisons were assessed. Fisher’s exact test was used to examine the significance of the association between the categorical data. All data analyses were performed using the JMP 13 statistical software . The study was approved by the Institutional Review Board in Kyushu University in Fukuoka, Japan .Supplementary Information.


Wnt/β-catenin is a developmental signaling pathway that plays a crucial role in driving kidney fibrosis after injury. Activation of β-catenin is presumed to be regulated through the posttranslational protein modification. Little is known about whether β-catenin is also subjected to regulation at the posttranscriptional mRNA level. Here, we report that insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) plays a pivotal role in regulating β-catenin. IGF2BP3 was upregulated in renal tubular epithelium of various animal models and patients with chronic kidney disease. IGF2BP3 not only was a direct downstream target of Wnt/β-catenin but also was obligatory for transducing Wnt signal. In vitro, overexpression of IGF2BP3 in kidney tubular cells induced fibrotic responses, whereas knockdown of endogenous IGF2BP3 prevented the expression of injury and fibrosis markers in tubular cells after Wnt3a stimulation. In vivo, exogenous IGF2BP3 promoted β-catenin activation and aggravated kidney fibrosis, while knockdown of IGF2BP3 ameliorated renal fibrotic lesions after obstructive injury. RNA immunoprecipitation and mRNA stability assays revealed that IGF2BP3 directly bound to β-catenin mRNA and stabilized it against degradation. Furthermore, knockdown of IGF2BP3 in tubular cells accelerated β-catenin mRNA degradation in vitro. These studies demonstrate that IGF2BP3 promotes β-catenin signaling and drives kidney fibrosis, which may be mediated through stabilizing β-catenin mRNA. Our findings uncover a previously underappreciated dimension of the complex regulation of Wnt/β-catenin signaling and suggest a potential target for therapeutic intervention of fibrotic kidney diseases.  Chronic kidney disease (CKD), characterized by progressive tissue fibrosis and gradual loss of kidney function, is becoming a major public health problem worldwide . CKD is Wnt/β-catenin is an evolutionarily conserved signaling pathway that is activated after tissue injury and plays a crucial role in driving tissue fibrosis . As a maInsulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) belongs to a highly conserved family of RNA-binding proteins, which also includes IGF2BP1 and IGF2BP2 –26. As RIn this study, we show that IGF2BP3 is induced in the kidneys of animal models and patients with CKD. Overexpression of IGF2BP3 impairs tubular cell integrity and promotes kidney fibrosis, possibly by stabilizing β-catenin mRNA. These studies identify IGF2BP3 as a major player in kidney fibrogenesis.We first assessed the expression of IGF2BP3 in various well-established animal models of CKD induced by unilateral ureteral obstruction (UUO), ischemia/reperfusion injury (IRI), adriamycin (ADR), and angiotensin II (Ang II), respectively. These models represent diverse etiologies that lead to renal failure and fibrotic lesions. As shown in We next examined IGF2BP3 localization in the fibrotic kidneys by using immunohistochemical staining. IGF2BP3 was undetectable in normal kidney but markedly upregulated in CKD . Strong https://doi.org/10.1172/jci.insight.162060DS1). IGF2BP2 localized in renal tubular epithelium as well  at 3 days after cisplatin or 1 day after IRI , suggestTo study the clinical relevance of these findings, we investigated IGF2BP3 expression in human kidney biopsies from patients with CKD. As shown in We further assessed fibrotic lesions in the kidney biopsies of patients with CKD after Masson’s trichrome staining and analyzed the relationship between IGF2BP3 and fibrotic lesions and kidney function in 25 cases of CKD. As shown in IGF2BP3 promoter by a bioinformatics approach. As shown in IGF2BP3 genes, which were perfectly matched with TBS consensus sequences  that selectively inhibited β-catenin–mediated gene transcription in HKC-8 cells. As shown in To delineate the role of IGF2BP3 in tubular cell biology, we examined the effect of its overexpression on kidney tubular epithelial cells. To this end, HKC-8 cells were infected with either control lentivirus (LV-Ctrl) or IGF2BP3-expressing lentivirus (LV-IGF2BP3) overnight and then incubated for 2 days as indicated. As shown in We found that overexpression of IGF2BP3 induced β-catenin and active β-catenin , suggestINK4A (a cellular senescence–related marker). To clarify whether these effects are mediated through IGF2BP3 regulation of β-catenin, HKC-8 cells were infected with either control lentivirus or IGF2BP3 lentivirus overnight, then incubated with ICG-001. As shown in We also examined the effect of IGF2BP3 on cell cycle arrest and cellular senescence, as they contribute to the pathogenesis of kidney injury and fibrosis. As shown in INK4A  or IGF2BP3 adeno-associated viral vector (AAV-BP3) into 6 sites of the left renal cortex for 12 weeks, as intraparenchymal AAV injections result in robust but relatively local transduction . ImmunosTo further investigate the role of IGF2BP3 in the pathogenesis of CKD, we used a mouse model of UUO, a widely used model with robust kidney fibrosis. To this end, mice were injected with AAV-Ctrl or AAV-BP3 at 12 weeks prior to UUO . WesternWe found that overexpression of IGF2BP3 promoted the expression of several fibrosis-related proteins and aggravated kidney fibrotic lesions. As shown in INK4A was upregulated in UUO mice, and overexpression of IGF2BP3 augmented these inductions.We also investigated the effect of IGF2BP3 on β-catenin signaling in vivo. As shown in To further confirm the role of IGF2BP3 in CKD, we sought to knock down IGF2BP3 by using an shRNA-mediated inhibition approach in vivo. Mice were injected intravenously with either Ctrl-shR or IGF2BP3-shR plasmids at 2 days after UUO . As illuWe next assessed the effects of IGF2BP3 depletion on fibrotic lesions after UUO. As shown in INK4A was upregulated in UUO mice, whereas knockdown of IGF2BP3 abolished the induction of these proteins. Immunostaining for β-catenin produced similar results  readers and promote the stability of their mRNA targets in an m6A-dependent manner , the 3′-UTR, or coding regions by recognizing specific RNA motifs , 43. Thet manner . However-catenin . In this-catenin . The preIn summary, the present study demonstrates that IGF2BP3, an RNA-binding protein, is upregulated in animal models and patients with CKD and critically involved in kidney fibrogenesis by activating β-catenin signaling. These findings underscore that regulation of β-catenin at the mRNA level, a process that was largely overlooked in the past, is an underappreciated dimension of the complex regulation of Wnt signaling. Although more studies are needed, our studies could provide a potential target for therapeutic intervention of fibrotic CKD.Male C57BL/6 mice and BALB/c mice, weighing about 20–25 g, were obtained from the Experimental Animal Center of Southern Medical University in Guangzhou, China. For the UUO model, C57BL/6 mice were used as described previously . BrieflyHuman kidney biopsy samples were obtained from diagnostic renal biopsies performed at the Nanfang Hospital, Southern Medical University, with written informed consent from the patients. Human normal kidney controls were obtained from nontumor renal tissues of patients who had renal cell carcinoma and underwent nephrectomy. Paraffin-embedded human kidney biopsy sections (3 μm) were prepared using a standard procedure and used for immunohistochemical staining. Quantification was assessed by a computer-aided point-counting technique.2. Serum-starved HKC-8 cells were treated by human recombinant Wnt3a protein  at varying dosages in the serum-free medium for various periods as indicated. In some experiments, HKC-8 cells were pretreated with ICG-001  at 10 μM for 1 hours, followed by incubation with vehicle or Wnt3a for an additional 2 days. Cells were then collected and subjected to various analyses.Human kidney proximal tubular cells (HKC-8) were provided by Lorraine C. Racusen . HKC-8 cells were grown in DMEM/Ham’s F12 medium supplemented with 10% fetal bovine serum (FBS). HEK293T cells were obtained from the American Type Culture Collection and cultured in DMEM supplemented with 10% FBS. These cells were cultured at 37°C in an atmosphere containing 5% COIGF2BP3 gene by infecting cells. The recombinant lentivirus vector expressing IGF2BP3, designated as LV-IGF2BP3, was constructed by Hanbio Biotechnology Co. Briefly, the full length of IGF2BP3 was inserted into a pHBLV-CMV-MCS-3xflag-EF1-ZsGreen-T2A-PURO vector. After validation by sequencing, lentivirus was packed, purified, and titrated. HKC-8 cells were infected with either LV-Ctrl (1.5 × 108 transduction units [TU]/mL) or LV-IGF2BP3 (3 × 108 TU/mL) overnight, then incubated for 2 days as indicated. The expression of the Flag-IGF2BP3 was assessed by Western blot analysis in HKC-8 cells. In some experiments, HKC-8 cells were transfected with pDel-β-cat using the Lipofectamine 2000 reagent  as previously reported  according to the procedures specified by the manufacturer.g at 4°C for 15 minutes, and concentration was determined with BCA protein assay . Gel electrophoresis was performed on reduced, denatured samples. After blotting onto PVDF microporous membranes  and blocking with 5% milk, membranes were incubated with primary antibodies and HRP-conjugated secondary antibodies. The protein bands were visualized by SuperEnhanced chemiluminescence detection reagents  and Kodak x-ray film. The primary and secondary antibodies used are listed in Whole-kidney homogenates and cells were prepared with RIPA buffer containing 1% NP-40, 0.1% SDS, 100 μg/mL PMSF, and 1% Halt Protease and Phosphatase Inhibitor Single-Use Cocktail  in PBS on ice. The supernatants were collected after centrifugation at 13,000Total RNA isolation was carried out using the TRIzol RNA Isolation Reagent . The first-strand cDNA synthesis was performed by using 2 μg of RNA in 20 μL of reaction buffer using a Reverse Transcription System kit (Promega). qRT-PCR was performed on ABI PRISM 7000 Sequence Detection System . The PCR reaction mixture was in a 25 μL volume including 12.5 μL 2× SYBR Green PCR Master Mix , 5 μL diluted reverse transcription product (1:10), and 0.5 μM sense and antisense primer sets. The mRNA levels of different genes were calculated after normalization with β-actin or GAPDH. The sequences of the PCR primer pairs used are described in 12 viral genomes [vg]/mL) or AAV-BP3 (1.7 × 1012 vg/mL) into 6 sites (10 μL at each site) of the left renal cortex with a glass micropipette (n = 5 per group): (i) sham+AAV-Ctrl, (ii) UUO+AAV-Ctrl, (iii) UUO+AAV-BP3, and (iv) sham+AAV-BP3. The detailed experimental design is presented in AAV9 vector was used as a gene transfer vehicle for overexpression of IGF2BP3 in the kidney. The recombinant AAV vector expressing IGF2BP3, designated as AAV-BP3, was constructed by Hanbio Biotechnology Co. Briefly, the full length of IGF2BP3 cDNA was inserted into a pHBAAV-CMV-MCS-3xflag-T2A-ZsGreen vector, and the accuracy of the inserted IGF2BP3 was validated by sequencing. AAV9 was packed, purified, and titrated. Male C57BL/6 mice were anesthetized and injected with AAV-Ctrl (1.9 × 10opipette . Pilot en = 5 per group): (i) sham, (ii) UUO+Ctrl-shR, and (iii) UUO+IGF2BP3-shR. The detailed experimental design is shown in Knockdown of IGF2BP3 expression in vivo was carried out by an shR-based inhibition. Briefly, IGF2BP3-specific shR expression plasmid (pLVX-IGF2BP3-shR) and empty vector (pLVX-shR) were administered into mice via tail vein injection, using an established hydrodynamics-based gene transfer approach, as described previously . The seqn = 5 per group): (i) sham, (ii) UUO injected with vehicle, and (iii) UUO injected with ICG-001. Mice were injected intraperitoneally with vehicle or ICG-001 at 5 mg/kg body weight once a day beginning from day 3 after UUO. Mice were sacrificed 7 days after UUO, and the kidney tissues were collected for various analyses.For assessing the effect of β-catenin signaling on IGF2BP3 expression in vivo, mice were divided into 3 groups . The levels of Scr were expressed as milligrams per deciliter.Paraffin-embedded mouse and human kidney sections (3 μm thickness) were prepared by a routine procedure , 49. TheRenilla reniformis luciferase driven under a thymidine kinase promoter  was also cotransfected for normalizing the transfection efficiency. The luciferase assay was performed using the Dual-Luciferase Reporter Assay System kit according to the manufacturer’s protocols . Relative luciferase activity (arbitrary units) was calculated as fold-induction over controls after normalizing the transfection efficiency.HEK293T cells were infected with either LV-Ctrl or LV-IGF2BP3 overnight and then incubated for 2 days as indicated. The HEK293T cells were then transfected with the TOPFlash luciferase reporter plasmid  using Lipofectamine 2000 reagent. A fixed amount (0.1 μg) of internal control reporter CTNNB1 and 18S rRNA, using equal volumes of RIP eluate for IgG and IGF2BP3 for cDNA production. 18S rRNA was used for normalization.HKC-8 cells at 80%~90% confluence were harvested by trypsinization and subjected to RIP. Briefly, RIP was carried out by using the RIP-Assay Kit  according to the manufacturer’s recommendations with normal rabbit IgG/RIP-certified IGF2BP3 antibody and protein A/G plus-agarose beads (Santa Cruz Biotechnology). Western blotting of a portion of washed RIP samples was performed to confirm specificity of IP. Following RNA isolation, RIP and input RNA were quantified. The IGF2BP3 RIP usually yielded more than 500 ng of RNA while IgG control was less than 50 ng. qRT-PCR was performed as previously described with the primers of CTNNB1 mRNA, control and IGF2BP3-depleted HKC-8 cells were incubated with actinomycin D  at 5 μg/mL. Cells were then collected at different time points as indicated, and RNA was isolated for qRT-PCR. β-Actin was used for normalization.For assessing the stability of CTNNB1 probes  were purchased from Boster. Paraffin-embedded kidney sections (3 μm) were fixed with 4% paraformaldehyde, and ISH was performed with a commercial kit (Boster) according to the procedures specified by the manufacturer.t test or 1-way ANOVA followed by the Student-Newman-Keuls test. Spearman’s (nonparametric) correlation analysis was used to assess the relationship between IGF2BP3 area and other variables. P < 0.05 was considered significant.All data examined were expressed as mean ± SEM. Statistical analyses of the data were performed using SPSS 19.0. Comparisons between groups were made by 2-tailed All animal studies were approved by the Animal Ethics Committee at the Nanfang Hospital, Southern Medical University. Studies involving human samples, for which written informed consent was provided for use, were approved by the Medical Ethics Committee at the Nanfang Hospital, Southern Medical University. All animal studies were in accordance with the US Public Health Service Policy on Humane Care and Use of Laboratory Animals.HF and Y Liu conceived the study. DS, HF, and Y Liu designed the experiments; DS, JS, Y Long, MZ, LL, JC, YX, HT, HZ, and XH performed the experiments; DS, HF, FFH, and Y Liu analyzed the data; FFH provided human kidney specimens; DS and Y Liu created the figures; DS and Y Liu wrote the manuscript; and all authors approved the final version of the manuscript.


Protein arginine methyltransferases (PRMTs) regulate protein biological activity by modulating arginine methylation in cancer and are increasingly recognized as potential drug targets. Inhibitors targeting PRMTs are currently in the early phases of clinical trials and more candidate drugs are needed. Flavokawain A (FKA), extracted from kava plant, has been recognized as a potential chemotherapy drug in bladder cancer (BC), but its action mechanism remains unclear.We first determined the role of a type II PRMT, PRMT5, in BC tissue samples and performed cytological experiments. We then utilized bioinformatics tools, including computational simulation, virtual screening, molecular docking, and energy analysis, to identify the potential use of PRMT5 inhibitors for BC treatment. In vitro and in vivo co-IP and mutation assays were performed to elucidate the molecular mechanism of PRMT5 inhibitor. Pharmacology experiments like bio-layer interferometry, CETSA, and pull-down assays were further used to provide direct evidence of the complex binding process.Among PRMTs, PRMT5 was identified as a therapeutic target for BC. PRMT5 expression in BC was correlated with poor prognosis and manipulating its expression could affect cancer cell growth. Through screening and extensive experimental validation, we recognized that a natural product, FKA, was a small new inhibitor molecule for PRMT5. We noticed that the product could inhibit the action of BC, in vitro and in vivo, by inhibiting PRMT5. We further demonstrated that FKA blocks the symmetric arginine dimethylation of histone H2A and H4 by binding to Y304 and F580 of PRMT5.In summary, our research strongly suggests that PRMT5 is a potential epigenetic therapeutic target in bladder cancer, and that FKA can be used as a targeted inhibitor of PRMT5 for the treatment of bladder cancer.The online version contains supplementary material available at 10.1186/s13046-022-02500-4. Bladder cancer (BC) is the most common type of urinary cancer worldwide, with high recurrence and mortality . AlthougUnbalanced methylation is gradually being accepted as a potential driver in human cancers . AlthougThe roles of PRMT enzymes in numerous diseases have spurred significant interest in targeting them pharmacologically . Among tKava has been used for treating inflammatory bladder diseases for more than 100 years, and chalcones are the main classes of compounds identified from kava extracts, including flavokawain A, B, and C , 19. TheIn previous studies, FKA could induce apoptosis in BC cells via the involvement of Bax protein-dependent and mitochondria-dependent apoptotic pathways . In addiThe present study revealed that PRMT5 was the most specific epigenetic regulator of all PRMT enzymes involved in BC and was highly correlated with the malignant characteristics of the disease. We identified FKA as a specific inhibitor targeting PRMT5, which could disrupt the binding of PRMT5 to histone H2A and H4, thereby inhibiting the symmetric arginine dimethylation level of histones. Our current findings suggest that PRMT5 is an attractive molecular target, and that FKA may be an effective small natural molecule drug for treating BC patients with abnormal PRMT5 activity.Bladder tumor cell lines, UMUC3 and T24, and urothelial cell line, SV-HUC-1, were acquired from the cell bank of the Chinese Academy of Sciences and cultured in high glucose DMEM or PRMI 1640 medium with 10% FBS . All reagents  were purchased from MedChemExpress, and various concentrations FKA were used to treat UMUC3 and T24 cells.Transient transfection was performed using LipoFiter 3.0  to introduce plasmid into cells. A PRMT5 expression cell line was constructed using lentivirus, and stable transfected cells were selected using puromycin.Surgically removed BC specimens and their adjacent normal counterparts were collected from the First Affiliate Hospital of China Medical University. The samples were stored at − 80 °C after collection. The study design was approved by the Institutional Ethics Committees at the First Affiliate Hospital of China Medical University. The patients’ medical records are provided in Cell lysates were extracted, standardized, and attenuated for electrophoresis. Proteins were separated using 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were then blocked at room temperature and incubated with the primary and secondary antibodies. The antibodies used are listed in the Antibodies were incubated with cell lysates prepared as described above in the presence of magnetic beads . Proteins were separated from beads by heating with SDS-containing loading buffer.Cells were fixed in 4% paraformaldehyde and permeabilized using 5% Triton X-100. Antibodies were incubated in 5% FBS overnight; DAPI staining was performed to visualize the nuclei. A confocal microscope  of 100 magnification was used for obtaining images.Briefly, HEK293T cells were transfected with PRMT5-Flag and treated with 40 μM Biotin–FKA for 4 h. The cells were then lysed in lysis buffer, and cell lysates were incubated with streptavidin agarose overnight at 4 °C. Next, the streptavidin agarose beads were denatured and re-suspended in SDS-containing loading buffer. The results were obtained using anti-PRMT5 or anti-Flag antibody.5 cells/mL in a growth medium and inoculated into a 24-well plate, to which 40 μM FKA was added, and then incubated at 37 °C and 5% CO2 for 1 h. The plate was then gradually heated from 40 to 68 °C, with temperature increments of 2 °C for 3 min each time. The plate was then immediately supplemented with the cell lysis and detection reagent. After 30 min of incubation at room temperature, the chemiluminescence was determined using a Multimode Microplate Reader. Data were collected, and melt curves were plotted in GraphPad Prism using a Boltzmann sigmoidal fit.CETSA was conducted as previously described by Lutz et al. . PlasmidAn Octet K2 system was used for conducting the binding experiments on the PRMT5 proteins with FKA samples. All PRMT5 samples were prepared in Octet buffer  at a concentration of 10 μM/L. Random-biotinylated FKA proteins were prepared at concentrations of 2, 3, and 4 μM. The hydrated streptavidin biosensors (ForteBio) first captured the biotinylated spike proteins for 60 s, and were then transferred to Octet buffer for 60 s to remove unbound proteins and provide the baseline. Then, they were immersed in diluted FKA samples for 120 s to provide the association signal, followed by transfer into Octet buffer to test for a disassociation signal for 240 s. The generated data were analyzed using the ForteBio Data Analysis software for correction and curve fitting.Total RNA was extracted using TRIzol reagent, cDNA was synthesized using the PrimeScript RT Master Mix kit . The PCR panel was obtained using the SYBR Premix Ex Taq kit , according to the manufacturer’s instructions. The primer sequences used are listed in the Cells were added into 96-well plates and imposed with certain conditions. CCK-8 reagent (GlpBio Tech.) was added with fresh medium after treatment, and absorbance was measured at 450 nm.g for 5 min, washed twice with PBS, and resuspended in the 400 μL 1× binding buffer; 5 μL of Annexin V-FITC and propidium iodide were added in turn and mixed. The treated cells were placed in the dark at room temperature for 5–15 min, after which flow cytometry analysis was performed.The apoptotic percentage of cells was assessed using an AnnexinV-FITC Apoptosis Detection Kit . Briefly, treated cells were centrifugated at 1000×6 cells per mouse, wild-type or PRMT5 knockdown UMUC3 cells, were injected subcutaneously, and FKA  was injected around the tumor in the wild-type group every 3 days. After 24 days of continuous observation, the mice were sacrificed, and the weights of xenografted tumors and mice were recorded.Four-week-old Balb/c nude mice were prepared at the China Medical University and experiments were conducted in accordance with the institutional guidelines approved by the Animal Care and Use Committee (IACUC Issue No. KT2022608). Approximately 5 × 10The Cancer Genome Atlas Program (TCGA) dataset was downloaded for mining in R . WGCNA aThe Glide docking method in Schrodinger software was used to predict the binding pattern of FKA and PRMT5 (PDB ID: 4X60), which generated a PDB file of the FKA-PRMT5 complex. Molecular dynamics simulations of the complex were performed using Amber14. The general AMBER force field (GAFF) was used for FKA, and the ff14SB force field was used for PRMT5. Molecular mechanics optimization and molecular dynamics simulation of all compounds were performed using the Sander or Pmemd procedures in Amber14. The binding free energy was calculated using the MM/PBSA method.The pattern diagram was drawn using the Figdraw software.t-test or one-way ANOVA. Statistical significance was considered at p < 0.05 .Statistical analysis was performed using GraphPad Prism software. All data were presented as the mean ± SD of at least three independent experiments, and all differences between the indicated groups were tested using the Student To assess whether PRMT family expression was associated with the development of BC, we performed an analysis of the PRMT family expression, which showed that most PRMTs were upregulated in tumor tissues compared with those in normal counterparts Fig. a. HoweveThis indicated that PRMT5 was a BC-prognosis associated arginine methyltransferase. Weighted gene co-expression network analysis on PRMTs identified the most correlated gene module with PRMT5 expression, and functional enrichment analysis showed that this module was correlated with the cell cycle and chromatin region, indicating the involvement of PRMT5 in regulating chromatin remodeling and alternative splicing Fig. a. After p-value, high PRMT5 expression shortened the median survival time by approximately 4 months, which may impede the effectiveness of atezolizumab  PRMT5 expression correlated with BLCA gene signatures in the TCGA dataset. (b) PRMT5 expression correlated with BLCA marker genes in the TCGA dataset. (c) Kaplan–Meier analysis of survival prognosis based on PRMT5 expression in Imvigor datasets. (d) IHC analysis of PRMT5 in different stage BC. (e) Western blot analysis of PRMT5 expression and histone methylation level in BC tissues and adjacent normal bladder tissues. Statistical analysis of protein expression was shown on the right side. Fig. S2. (a) Cell viability assay performed by treating FKA in urothelial cell SV-HUC-1. (b) Cell viability assay performed after treatment with FKB, and FKB IC50 were calculated in T24 (left) and UMUC3 (right). Data are represented as mean ± SD in three replications. (c) Cell viability assay performed after treatment with FKC, and FKC IC50 were calculated in T24 (left) and UMUC3 (right). Data are represented as mean ± SD in three replications. (d) PRMT5 expression changed with different concentrations of FKB treatment times in T24 (upper) and UMUC3 (lower). (e) PRMT5 expression changed with different concentrations of FKC treatment times in T24 (upper) and UMUC3 (lower). (f) Different PRMT expression changed with different concentrations of FKA in T24 (left) and UMUC3 (right). Fig. S3. (a) Cell apoptosis measured by knocking down PRMT5 expression, FKA treatment, and supplied FKA in PRMT5 overexpressed UMUC3 using flow cytometry. (b) Apoptosis rates for replicated assays were counted. (c) Cell viability assay performed after treatment with FKA or PRMT5 shRNA combined with cetuximab in T24 (left) and UMUC3 (right). Data are represented as mean ± SD in five replications. (d) Functional pathway enrichment of predicted FKA downstream targets. (e) Bladder cancer regulon genes changes after supplying FKA in UMUC3 measured by PCR, and the fold changes were standardized and normalized by log10. Fig. S4. (a) Bio-Layer Interferometry detecting the combination and dissociation constants of FKA and human recombinant PRMT5. (b) Fluorescent report CETSA assay confirmed that 25 μM FKA treatment could inhibit PRMT5 degradation when heated, thus enhancing PRMT5 stability in UMUC3. (c) Fluorescent report CETSA assay performed for PRMT5 expression when treated with FKA in wild-type and Y304A/ F580A mutated PRMT5 in UMUC3 cells. (d) Conservation of PRMT5 Y304 and F580 in different species.