ATM and TP53, which are accumulated as therapy resistance emerges and the disease progresses, consistent with our OncoPrint results that ATM and TP53 alterations were most frequent in relapsed/refractory (R/R) MCL. We demonstrated that protein arginine methyltransferase-5 (PRMT5) was upregulated in R/R MCL, which predicted a poor prognosis. PRMT5 inhibitors displayed profound antitumor effects in the mouse models of MCL with mutated ATM and/or TP53, or refractory to CD19-targeted CAR T-cell therapy. Genetic knockout of PRMT5 robustly inhibited tumor growth in vivo. Co-targeting PRMT5, and ATR or CDK4 by using their inhibitors showed synergistic antitumor effects both in vitro and in vivo. Our results have provided a rational combination therapeutic strategy targeting multiple PRMT5-coordinated tumor-promoting processes for the treatment of R/R MCL with high mutation burdens.Constant challenges for the treatment of mantle cell lymphoma (MCL) remain to be recurrent relapses and therapy resistance, especially in patients harboring somatic mutations in the tumor suppressors  MCL is an incurable and aggressive subtype of non-Hodgkin B-cell lymphoma , 2. MajoATM) and TP53 genes [CCND1 to the IGH gene, a hallmark of MCL, leads to the overexpression of cyclin D1 [CDKN2A and MTAP on chromosome 9p21.3 are frequently co-deleted in MCL, especially in ibrutinib (IBN)-resistant tumors [CDKN2A gene encodes for the tumor suppressor p16INK4A protein, a negative regulator of CDK4/6. Loss of p16INK4A leads to sustained activation of CDK4/6, resulting in uncontrolled proliferation [MCL frequently accompanies with a high degree of genomic instability and multiple somatic mutations in DNA damage repair (DDR) pathways, especially in ataxia-telangiectasia-mutated  [PRMT5 is a type-II arginine methyltransferase and forms a tight hetero-octameric complex with its substrate-binding partner MEP50 (encoded by WDR77) . Along wWDR77) . PRMT5 sWDR77) , transcrWDR77) , 20, andWDR77) . ImportaWDR77) , indicatWDR77) , 23, 24 WDR77) . PRMT5 uWDR77) , making WDR77) –29.ATM is the most frequently mutated gene in the MCL mutational landscape [RB1 and TP53, the ATM kinase plays a central role in the cellular response to DNA double-strand breaks. ATM gene defects in MCL patients frequently resulted in a complete inactivation of ATM [TP53 mutations have been frequently observed in MCL with blastoid/pleomorphic histology [ATM abrogates p53 activation in response to DNA damage, allowing the cells with unrepaired DNA to escape from p53 surveillance [ATM and TP53, we screened numerous promising compounds targeting DDR, p53 signaling, and cell cycle, including PRMT5 inhibitors, for their cytotoxicity on MCL cell lines with different genetic backgrounds.andscape . Coordinn of ATM . TP53 muistology . Loss ofeillance . To explATM and TP53 genes in most R/R patients with MCL, including those with intrinsic or acquired resistance to ibrutinib or CD19-targeted CAR T-cell therapy. MCL cells with frequent mutations in genes including ATM or TP53 are sensitive to PRMT5 inhibition or depletion. Using our preclinical cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models, our study has identified a rational combination therapeutic strategy which acts to restrain the dysregulated DNA damage response and cell cycle progression in MCL.In this study, we presented evidence to demonstrate frequent genetic alterations in The MCL cell lines JeKo-1, Mino, Z-138, Maver-1, Rec-1, JVM-2, JVM-13, UPN1, HBL-2, SP-49, and the human lymphoblastoid cell line (LCL) were obtained from the American Type Culture Collection. The Granta-519 cell line was obtained from the Leibniz-Institute DSMZ. Peripheral blood, bone marrow, biopsies, and apheresis specimens were collected from patients with MCL after consent and approval from the Institutional Review Board at the University of Texas MD Anderson Cancer Center.5 cells per well in 24-well plates and treated with indicated inhibitors for 5 d. The live cells were counted each day.Cell cytotoxicity screening was conducted as described previously , using C5 cells per well in 24-well plates and incubated with indicated inhibitors for various periods of time, then stained with annexin-V (BD Biosciences) and propidium iodide (Invitrogen). Apoptosis was determined using flow cytometry on NovoCyte Flow Cytometer (ACEA Biosciences), and the data were analyzed with FlowJo v10.Cells were seeded at 2 × 10Cell cycle analysis was done as previously described . The celROS level in MCL cells was measured as described previously , using HWestern blotting was performed as described previously . The antTotal RNA was extracted from treated MCL cell lines using Qiagen RNeasy Mini Kit. First-strand cDNA was synthesized using the iScript Reverse Transcription Supermix. cDNA was amplified with MDM4 exon-specific primers as described elsewhere .Total RNA was extracted from treated MCL cell lines and cDNA was prepared as described above. Expression of specific mRNAs was determined by qPCR using the SYBR green supermix (Bio-Rad) as described elsewhere .Selected specimens from patients with MCL were subjected to whole-exome sequencing (WES) and the raw output from Illumina was analyzed for somatic mutations, functional annotation, and expression of mutant alleles, as described previously .Immunofluorescence staining was performed as previously described . Images P values were determined by chi-square test (F), or Student’s t test using the Prism software.Tumor microarrays (TMA) were obtained from the MD Anderson Tissue Bank after consent and approval from the Institutional Review Board at the University of Texas MD Anderson Cancer Center and used for immunohistochemistry as described elsewhere . The folMetabolites extracted from either ibrutinib-sensitive or -resistant MCL cell lines were subjected to targeted metabolomics analysis for nucleic acids and amino acids and analyzed as described previously .Gene knockout in MCL cell lines was achieved using CRISPR/Cas9 genome editing, as described previously .2 × 0.5, and tumor growth was monitored until humane endpoints.All experimental protocols were approved by the Institutional Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center. NSG mice at 6–8 weeks old were injected subcutaneously with MCL cell lines for CDX mouse models, or patient-derived MCL cells for PDX. The mice were treated when the tumors became palpable. Tumor volumes were measured on specified days and calculated using the formula: volume = length × widtht test, two-way ANOVA, or nonlinear regression analysis. Overall survivals were calculated with the Kaplan–Meier method and compared with the log-rank test.All assays were performed in triplicate and expressed as mean ± SEM or SD. The statistical significance of differences was determined by Student’s ATM and TP53 alterations were the most frequent in this 34 MCL patients   and 60% (TP53) were missense mutations, with the remaining annotated as frameshift, deletion, truncation, nonsense, and multiple mutations, which are predicted to lead to loss or gain of functions to the genes. Noteworthy, among the top 10 most frequently mutated, KMT2D, NSD2, RB1, CCND1, and CDKN2A are known to play important roles in the lymphomagenesis and disease progression of MCL. We next performed a meta-analysis on the public datasets from multiple MCL studies containing a total of 2008 evaluable MCL samples at either baseline or disease progression [ATM and TP53 at disease progression are apparently higher than that at baseline  datasets for the mutational frequency of genes in 34 MCL patient specimens from two studies , 33. Amoly) Fig. . Among tgression  and founine Fig. . Incorpones Fig. .Fig. 1MCATM, TP53, and CDKN2A  and/or CDKN2A-deficient  MCL cell lines are sensitive to PRMT5 inhibitor GSK3326595  in ATM-deficient and/or CDKN2A-deficient cell lines -modified proteins and H4R3me2s (histone H4R3 dimethyl symmetric), an arginine methylated substrate of the enzyme, in cells treated with the inhibitors  were substantially upregulated in ibrutinib-resistant MCL cells  and deletion or mutations on both ATM alleles with heterozygous deletion of TP53 (Granta-519)  than in ATM-proficient JeKo-1 (157)  Fig. . This re57) Fig. , along w57) Fig. . To vali57) Fig. .Fig. 4PRActivation of p53 is essential for functional ATM/p53 signaling. In addition to phosphorylation by ATM at multiple sites that stabilizes p53, p53 accumulation is complexly modulated by other post-translational modifications, including ubiquitination by MDM2 and MDM4, two negative regulators of p53, leading to its degradation. The MDM4 alternative splicing events including exon skipping are considered as sensors of PRMT5 depletion and defects in the constitutive splicing machinery . To inteTP53 status in MCL, as the drug also induced massive apoptosis in TP53-deficient JeKo-1 cell  and 0.49–2.5 μM (LLY-283)  in clinical specimens from MCL patients who were resistant to ibrutinib or the CAR T-cell therapy  with different ice Fig. , accompaice Fig. . Importawth Fig. , with dewth Fig. . Of noteATM and/or TP53 alterations, we performed dose-response cytotoxicity analysis of PRMT5 inhibitors in combination with DDR protein inhibitors. Combination treatment with PRMT5 and PARP inhibitors displayed a synergistic cytotoxic effect in ATM-deficient Granta-519 and Maver-1 cells  and -proficient (JVM-2) MCL cell lines (Fig. TP53 mutations and deletion of CDKN2A and treated the mice with either GSK3326595, abemaciclib, or in combination. Each inhibitor remarkably attenuated tumor growth, whereas the combination treatment exhibited a more profound tumor suppression (Fig. CDKN2A.RNA-seq analysis of specimens from MCL patients also revealed that CDK4 mRNA was significantly upregulated in ibrutinib-resistant MCL cells Fig. , in lineMCL Fig. . To examlls Fig. . This renes Fig. . To asseion Fig. , with noion Fig. . Westernion Fig. . These dATM and TP53, persist in MCL and continue to accumulate as the disease evolves [CDKN2A and MTAP on chromosome 9p21.3 [CDKN2A, CDK4 loses its negative regulator and therefore over-activated. As a potent driver of cell malignancy, PRMT5 is involved in multiple cell survival and proliferation pathways including DDR, p53 activation, cell cycle regulation, and c-Myc in tumor cells through protein arginine methylation and downstream effects such as RNA splicing regulation. Examination of our clinical specimens confirmed that PRMT5 was significantly upregulated in MCL, especially in ibrutinib-resistant patients. High expression of PRMT5 predicted poor prognosis, presenting as another potential therapeutic target for MCL.Among those derailed signaling, somatic mutations in DNA damage repair pathways, especially  evolves . Anothere 9p21.3 . WithoutATM, TP53, and/or CDKN2A-deficient CDX or PDX models, indicating that they are more dependent on PRMT5 which could be exploited as a potential therapeutic target for R/R MCL with heavy mutation burdens.Although our data could not explicitly conclude that the sensitivities of MCL cells to PRMT5 inhibition are exclusively dependent on the particular genetic status, the PRMT5 inhibitors exerted more potent cytotoxicity in ATM and TP53 mutations. Using CDX models, we present evidence that the combination of PRMT5 inhibitors with either DDR or cell cycle inhibitors significantly increased the anti-lymphoma potency in MCL with ATM and/or TP53 lesions, attributing to the intrinsic interaction between PRMT5 and DDR pathways. PRMT5 promotes homologous recombination repairing during DNA damage via guiding alternative splicing of the key DNA repair protein TIP60 [ATM deficiency could be more sensitive to PRMT5 inhibition than those with proficient ATM. In line with this hypothesis, we found that ATM-deficient PDX tumors (Fig. ATM deletion is required to clarify the relationship between PRMT5 and ATM. PRMT5 inhibitors have been reported less efficient in p53 knockout cells [In this work, we primarily aimed at identifying potential therapeutic strategies for R/R MCL featured with frequent in TIP60 . Our datin TIP60  also unrors Fig.  as well ors Fig. , are senut cells . We alsoATM, TP53, and CDKN2A. Considering the synergistic effect of the inhibition of both PRMT5 and DDR or cell cycle effector molecules, we anticipate our preclinical study has the translational potential to lead to new clinical trials for MCL.In conclusion, our results provide a rational combination therapeutic strategy by targeting PRMT5 in R/R MCL patients featured with frequent mutations in Supplementary Figures and TablesTable S3


Mesenchymal stem cells (MSCs) and their derivant are among the promising treatments for intrauterine adhesion (IUA); they have been reported to repair the endometrial injury by proliferating endometrial cells. However, the signal pathways involved are not clear. This study investigated the role of human umbilical cord mesenchymal stem cell-derived conditioned medium (hUCMSC-CM) in relieving IUA to find out whether Wnt/β-catenin signaling was measured by Western blots, fluorescent staining, and T-cell factor/lymphoid enhancer factor (TCF/LEF) luciferase. A Wnt inhibitor (XAV393) was used to inhibit the proliferation effect of hUCMSC-CM in EECs. Wnt5a expression in hUCMSC was measured by Western blots and fluorescent staining, and Wnt5a in hUCMSC-CM was detected by enzyme-linked immunosorbent assay (ELISA), to further clarify the mechanism.  After endometrial epithelial cells (EECs) were treated with hUCMSC-CM, their proliferation and migration were measured by the CCK8 assay and the scratch assay. The activation of Wnt/β-catenin, c-myc, and cyclin D1 increased in EECs after being treated with hUCMSC-CM. Moreover, hUCMSC-CM was found to promote β-catenin delivery into nuclei by Western blot and fluorescent staining; meanwhile, the inhibitor (XAV393) could restrain this process and inhibit the effect of hUCMSC-CM on EEC proliferation. Wnt5a was detected in hUCMSCs and hUCMSC-CM, which might be a potential therapeutic target.  As shown by the CCK8 assay, hUCMSC-CM promoted proliferation and migration of EECs. The expression of β-catenin signaling, and Wnt5a might be a potential activator. This would be one of the activating signal pathways in the MSC-related treatment of IUA. This study demonstrated that hUCMSC-CM promoted human endometrial cell proliferation through Wnt/ Intrauterine adhesion (IUA), also known as Asherman's syndrome (AS), is a complex gynecological pathology mainly caused by the intrauterine operation, infection, and congenital anomaly of the uterus. It can be characterized by fibrous, connective tissue bands in the uterine cavity, with or without glandular tissue , leadingβ-catenin signaling is critical in estrogen-mediated uterine development 04). The donors also provided informed consent, in accordance with the Declaration of Helsinki. Human EECs were purchased from Procell Life Science & Technology Co., Ltd., Wuhan, China. The authors had no conflict of interest.2 at 37°C. The medium was changed every day. Next, the large particle pieces were removed, and the adherent cells were subcultured using 0.25% trypsin. The hUCMSCs from the third to the fifth passages were used for further experiments [The umbilical cords were washed with phosphate buffer saline  containing 5% penicillin/streptomycin (P/S). Wharton's jelly was cut into small pieces and exposed to collagenase type IV solution (1 : 1 in PBS) for 1 h. They were cultured in Dulbecco's modified Eagle's medium , supplemented with 10% fetal bovine serum (Gibico) and 1% P/S, and incubated in a humidified atmosphere containing 5% COeriments .The hUCMSCs were verified by a flow cytometer  after staining with the specific antibodies, including CD 34, 44, 45, 73, 90, and 11b conjugated with FITC and APC, respectively.The hUCMSCs (third passage) were washed with PBS three times until they were 70–80% confluent, then they were preincubated with serum-free fresh DMEM/F12 for 48 h. The CM was collected, centrifuged at 1,000 rpm for 20 min, and filtered through a 0.22 um filter.Wnt5a in the CMs was measured by the enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions .4 cells/well) after being cultured with hUCMSC-CM or serum-free DMEM/F12 for 48 h. The CCK-8 reagent  was then added to the plates, which were incubated in a humidified atmosphere containing 5% CO2 at 37°C for 3 hours, and each sample was measured at 450 nm.EECs (P3) were seeded in 96-well plates , followed by a reverse transcription using a cDNA synthesis kit . The cDNA was followed by a real-time quantitative polymerase chain reaction (PCR) using the KAPA SYBR® FAST Master Mix  with a primer. Each gene expression level was normalized, with GAPDH used as a housekeeping control. The primer sequences were as follows: Wnt5a, forward 5′-CTCGCCATGAAGAAGTCCA-3′ and reverse 5′- TACCTAGCGACCACCAAGAA -3′; cyclin D1, forward 5′- GCTGCGAAGTGGAAACCATC -3′ and reverse 5′- CCTCCTTCTGCACACATTTGAA -3′; c-myc, forward 5′- GTCAAGAGGCGAACACACAAC -3′ and reverse 5′- TTGGACGGACAGGATGTATGC -3′; CTNNB1, 5′- GGTGGGCTGCAGAAAATGGTT -3′ and reverse 5′- GATGGCAGGCTCAGTGATGTCTTC -3′.Cells were lysed with RIPA lysis buffer  containing 1 × PMSF  to extract total protein.μg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes . The membranes were incubated with antibodies against β–catenin , the following antibodies used the same dilution buffer), C-myc (1 : 1000), Cyclin D1(1 : 1000), GAPDH (1 : 5000), Wnt5a (1 : 1000), β-tubulin (1 : 2000) , and Histone (1 : 2000)  at 4°C overnight and then incubated with corresponding horseradish peroxidase- (HRP-) conjugated second antibodies (1 : 1000)  at 37°C for 1 h. The bands were visualized with enhanced chemiluminescence . The intensity of each band was calculated using Image J software .Nuclear and cytoplasmic proteins were extracted separately with the Nuclear and Cytoplasmic Protein Extraction Kit . Protein lysates (20 β-catenin antibodies (1 : 200)  at 4°C overnight in a humidified atmosphere. They were then incubated with Alexa Fluor488-conjugated secondary antibodies (1 : 500)  for 1 h, and the nucleus was stained with DAPI  for 5 min. Immunofluorescence staining was imaged using fluorescence microscopy .After treatment, the cells were fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 in PBS, and blocked in 1% bovine serum albumin for 1 h. Then, the cells were incubated with primary μg of either TOPflash or FOPflash vector  and 0.1 μg of pRL-TK (Renilla-TK-luciferase vector) as a control using Lipofectamine 3000 (Invitrogen). Some cells were subsequently treated with hUCMSC-CM, while the control group was treated with the serum-free DMEM/F12 for 48 h. The cells were then lysed, and the luciferase activities were measured using the Dual-Luciferase Reporter Gene Assay Kit , according to the manufacturer's instructions. Firefly luciferase activity was normalized against the Renilla luciferase activity, and the TOP/FOP ratio was used as a measure to evaluate the activation of the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription.The cells were seeded into a 24-well plate at a density of 50,000 cells per well. The cells were cotransfected with 0.5 5 cells per well. After cell adherence, scratches were made with a sterilized P200 pipette tip (500-μm width), and the cells were cultured in hUCMSC-CM or serum-free DMEM/F12 after washing with PBS. After 0 h, 48 h, and 96 h, the scratches were photographed by microscopy  and measured manually with Image J software. The data were reported as the ratio of migration relative to the control.The cells were seeded in 6-well plates at 5 × 10t-test, and a P value < 0.05 indicated significant differences.The results are expressed as mean ± SEM. All experiments were repeated three times with independent cultures. Statistical significance was assessed using Student's Flow cytometry was used to detect the positive markers  and the negative markers  of the MSCs to identify the hUCMSCs. The results demonstrated that the positive markers were highly expressed, but the negative markers were not expressed .The CCK8 assay was performed to investigate the effect of hUCMSC-CM on endometrial cells proliferation. The results showed that hUCMSC-CM promoted the proliferation of EECs . The scrAfter being treated with hUCMSC-CM for 48 h, the mRNA expression of CTNNB1 in EECs was detected. The results of the CTNNB1 gene expression were significantly higher in the hUCMSC-CM group than in the control group (treated with serum-free DMEM/F12) , and theAfter being treated with hUCMSC-CM for 48 h, the mRNA expression of downstream gene CMYC and CYCLIND1 in EECs was detected. The CYCLIND1 gene expression was significantly higher in the hUCMSC-CM group than that in the control group (treated with serum-free DMEM/F12), while C-MYC showed no statistical difference . Both haβ-catenin protein levels in nucleus and cytoplasm were detected, respectively. In the hUCMSC-CM groups, the β-catenin protein levels were higher in nucleus than those of the control groups  to hUCMSC-CM inhibited the proliferation of endometrial cells in the control group compared with the hUCMSC-CM group  receptor and coreceptor and low-density lipoprotein receptor-related protein 5/6 (LRP 5/6), initiating β-catenin nuclear translocation. β-Catenin is an unstable protein and is strictly regulated in the cytoplasm. In the absence of a Wnt ligand, intracellular β-catenin is targeted by a degradation complex. The complex consists of tumor suppressor protein (APC), scaffold protein Axin, and two kinases: tyrosine kinase 1α (CK1α) and glycogen synthase kinase 3β (GSK-3β). CK1α and GSK-3β phosphorylated β-catenin N-terminal serine and threonine, phosphorylated β-catenin recognized by transducers, which are part of the ubiquitin ligase complex, then β-catenin was degraded after ubiquitination. When the Wnt ligand binds to Fzd and LRP5/6, it induces phosphorylation of disheveled protein (DVL/DSH), recruits Axin protein, and then disintegrates the degradation complex. β-Catenin accumulates in the cytoplasm and translocates into the nucleus, where it binds to the TCF/LEF to regulate the target gene of the Wnt pathway [In the canonical Wnt signaling pathway, also known as the Wnt/ pathway .β-catenin was expressed in endometrial tissue in the middle and the late proliferation phases and increased in the nucleus of proliferative endometrial cells stimulated with high-dose estrogen without progesterone. In the secretion phase, when progesterone increases and estrogen decreases, the nucleus β-catenin decreases. Hou et al. [β-catenin in the nucleus of the endometrial epithelium after giving exogenous estrogen to mice. Furthermore, they delivered adenoviral expressed SFRP2, the Wnt signaling inhibitor, in mice and observed that estrogen-induced promotion of EECs proliferation was inhibited. Gunin et al. [β by giving LiCl, activator of the Wnt/β-catenin pathway, and successfully simulated the proliferation of endometrium in mice.The canonical Wnt signaling pathway plays a role in endometrial cell proliferation. Nei and his colleagues  collecteu et al.  detectedn et al.  inhibiteβ-catenin signaling was involved. By using the Wnt pathway activator (Wnt3a CM) and the Wnt inhibitor (XAV939 and IWP-2), the participation of β-catenin signaling in the self-renewal of eMSCs was confirmed. The high expression of Wnt5a was detected in uterine myocytes. The introduction of recombinant Wnt5a can stimulate eMSCs colony formation, while the Wnt5a antibody inhibited the colony formation. It is inferred that uterine myocytes are the activated microenvironment components of eMSCs, and the Wnt5a/β-catenin signaling pathway is an important signaling pathway to activate eMSCs. Bukowska et al. [Wnt signaling molecules can also activate eMSCs and participate in endometrial periodic regeneration. Cao et al.  found tha et al.  found thβ-catenin signaling [β-catenin, indicating that the component in the hUCMSC-CM might activate Wnt/β-catenin signaling, upregulate the expression of c-myc, and regulate the cell cycle of EECs, promoting the cells' proliferation.C-myc and cyclin D1 are the downstream genes of Wnt/ignaling , 27; theignaling  and moduignaling . It can ignaling . In thisβ-catenin signaling was activated by hUCMSC-CM, participating in EEC proliferation. Wnt genes were found expressed in hUCMSCs; among them, a relatively high mRNA expression of the Wnt5a was detected [β-catenin signaling might be involved.Many proteins are responsible for the development and regeneration of the endometrium, of which Wnt, c-kit (CD117), Oct-4, CD34/KLF4, and Musashi-1 are the best classified . In thisdetected . Consistβ-catenin pathway has been examined in vitro; in vivo studies need to be conducted in future work.Regarding the limitations of our work, first, the complex signal network and cross-talk are not discussed in this article. Second, other Wnt-ligand proteins, such as Wnt7a and Wnt4, were reported to play an essential role in endometrial development and regeneration of reproductive tracts; however, whether or not they relate to IUA treatment has not been addressed in this paper. Third, the Wnt/β-catenin signaling, and Wnt5a might be an activator. This would be one of the activating signal pathways in the MSC-related therapy of IUA.Our study demonstrated that hUCMSC-CM promoted human endometrial cell proliferation through Wnt/


However, achieving the high yield, low price and high activity required for commercial viability remains challenging. In this review, we first present a brief introduction, looking at the sources, characteristics, catalytic process, and applications of glucose oxidase. Then, the predictive structures of glucose oxidase from two different sources are comparatively discussed. We summarize the inhibitors of glucose oxidase. Finally, we highlight how the production of glucose oxidase can be improved by optimizing the culture conditions and microbial metabolic engineering.Glucose oxidase, which uses molecular oxygen as an electron acceptor to specifically catalyze the conversion of β- In the oxidation half-reaction, FADH2 is oxidized to FAD, and molecular oxygen accepts electrons and protons to form H2O2 [Aspergillus niger are shown in Aspergillus niger and Penicillium amagasakiense GOX, respectively [In the catalytic process, GOX uses molecular oxygen as an electron acceptor to specifically catalyze β- carrier ,25,26. dcatalase . This prorm H2O2 ,28. The Staphylococcus aureus, Salmonella infantis, Clostridium perfringens, Bacillus cereus and Listeria monocytogenes) prevention [GOX is considered to be an important enzyme that can be applied in bioelectrochemistry, food, pharmaceutical, medical diagnostic, and feed field. The use of GOX in biosensors and biofuel cells has previously been fully discussed in terms of clinical and personal use , glucoseevention , chemicaevention , browninevention ,35,36. Cevention  and Wangevention . A recenevention . Hence, Heliothis viriplaca and Citrobacter freundii were described and compared with a known structure of Aspergillus niger GOX. The inhibitors of GOX are introduced through an extensive assessment of the literature. Related strategies for enhancing GOX production and activity are highlighted in the discussion.In this review, the function, production, and properties of GOX summarized in previous reviews are briefly introduced ,4,24,40.Penicillium and Aspergillus have been well-studied. They consist of two identical subunits, and each monomer has two distinct domains: one is mainly a β-sheet, and is tightly but not covalently bound with FAD; the other is supported by four α-helices and an antiparallel β-sheet, and binds to the substrates  and 1.8 Å resolution (PDB 1GPE), respectively [Aspergillus niger and Penicillium amagasakiense has been analyzed in detail, but the structures and analysis of GOX from other strains are still lacking. For studying GOX from different strains, ProtParam (https://web.expasy.org/protparam/ (accessed on 16 February 2022)) can be used to make predictions about some of their properties, and the Phyre2 web can be employed to predict their protein structures [Aspergillus niger, the GOX structures derived from fungi do not have so many differences, while the structures from insects and bacteria are quite different. Here, Aspergillus niger sequences (PDB 1CF3) and other sequences were aligned using ClustalW ). The results showed that the GOX protein structures from Aspergillus , Penicillium  and Talaromyces  have a high similarity (60–80%), while the other species have a low similarity (15–30%). Although the amino-acid sequence alignment and structures of GOX from some fungi and insects have been described and discussed by Bauer et al. [Citrobacter freundii and Heliothis viriplaca has not been fully overviewed. Citrobacter freundii is the only bacterial strain that has been reported in Chinese regarding GOX production, and the sequence has been submitted to the NCBI database [The crystal structure of GOX from ectively . Other eructures . Comparedatabase .Citrobacter freundii (GenBank: QGP74835.1) and Heliothis viriplaca (GenBank: AMR44226.1) with a 12% and 20% similarity in their amino-acid sequence, respectively, compared with the well-known structure from Aspergillus niger. Their structures and substrate binding pockets are greatly varied. Despite this, it has been reported that they showed the same feature when specifically catalyzing β-d-glucose with high or low efficiency. As shown in Heliothis viriplaca is very close to the expected one, with an analogous binding pocket and an approximative binding free energy; while their residues remain independent. It is believed that residues in loops, either remote or near the active site, can affect substrate recognition and catalysis by conformational changes and reshaping the binding pocket [Aspergillus niger and Heliothis viriplaca performed differently. Meanwhile, the predictive structures of GOX from Citrobacter freundii indicates more obvious differences, not just from the active site but also from the binding pocket  indicates that it has a lower affinity to the surrounding amino-acid compared with the other two GOX. This may partially give explanatory notes to the fact that few GOX from bacteria has been in-depth investigated.g pocket . This cog pocket A,C. The These results pinpoint the discrepancies between GOX from different sources, especially from animals and bacteria. The lack of a precise crystal structure and knowledge of the molecular mechanism of these GOXs further impedes the full comprehension of different types of GOX. Given the ongoing improvements in accurate protein structure prediction (such as AlphaFold) and structure analysis , we can +, Co2+, Hg2+) [2O2, hydroxylamine, hydrazine, phenylhydrazone, dimedone, sodium bisulfate, guanidine hydrochloride, urea, sodium dodecyl sulfate) [+ can inhibit the oxidation of GOX by competing with molecular oxygen and combining with amino acid residues in or near the FAD moiety to reduce enzyme activity [2+ dilutes enzyme activity by inhibiting the oxidation of the reduced enzyme and weakening the binding of D-glucose to the enzyme with steric hindrance [+, Co2+, Hg2+ at 1 mM concentration exhibited obvious inhibitions of GOX activity [Inhibitors can cause a reduction in enzyme activity or enzyme inactivation. Enzyme inhibitors are selective and can be an impetus to elucidating enzymes’ mechanism of action and metabolic pathways. Only by understanding the inhibitor’s mode of action can we come up with a solution to provide GOX with better activity and stability in the industry. GOX activity can be inhibited by metal ions   and chemsulfate) ,50. Ag+ activity . Hg2+ diindrance . The resactivity .2O2 is a competitive inhibitor of GOX [2O2 [Hr of GOX , and canr of GOX . A totalGOX [2O2 . In addiGOX [2O2 . Thus, tIn the of GOX production using microorganisms, high yields are aided by the provision of an appropriate culture medium and conditions. The components in the medium play important roles in the growth of microorganisms and biosynthesis of biochemicals. The carbon sources are responsible for the growth and metabolism of microorganisms, and provide cell’s carbon framework and provide the energy required for cell life activities. Meanwhile, the nitrogen source contains proteins and nucleic acids for living organisms. The study of different carbon and nitrogen sources that affected the yield and activity of GOX proposed that high levels of GOX were obtained using molasses as carbon source and peptone as nitrogen source . A studyIn addition, the efficiency of microbial GOX production is usually dependent on temperature, pH, and dissolved oxygen. Most strains used for GOX production display an optimum temperature of 25–37 °C. The pH mainly affects the structure of bacterial cell membranes, the absorption and metabolism of nutrients, the activity of enzymes, and the direction of the metabolism. As reported in the literature, the optimum pH value for GOX production strains was in the range of 6.0–7.0 . In addiAspergillus niger by UV irradiation resulted in a 1.57-fold and 1.98-fold increase in the in vitro and in vivo enzyme activities of the obtained mutant glucose oxidase, respectively [Aspergillus niger to change, and the enzyme activity of the mutants increased by 282% and 202%, respectively. It was believed that the random mutagenesis of Aspergillus tubingensis using nitric acid and UV irradiation can screen for some mutants, leading to a significant yield improvement [Aspergillus niger and used them for protoplast fusion in intraspecific hybridization, resulting in fusants with higher activity than the original strain (from 114.5 to 332.1%).Mutagenesis is a primary and efficient way of engineering strains to change their performance, usually followed by a special environmental conditions screening for the desired mutations . Common ectively . Chemicaectively . A studyectively  revealedrovement . Meanwhirovement  selectedcat for glucose (from 69.5/s to 137.7/s) [Aspergillus niger [In addition, the further study of these mutations can provide information for rational design at the molecular level to promote GOX production . The dir137.7/s) . It has us niger . Throughus niger .Site-directed mutagenesis has the advantages of being more purposeful, faster, and more efficient, but the site-directed mutagenesis of targeted residues is often labor-intensive and time-consuming, and the effect of mutations on protein function is always unpredictable . HoweverAspergillus and Penicillium [Heterologous expression is an important molecular technique for the expression of foreign gene proteins by bacteria, yeast, animal cells, and plant cells to enhance production efficiency. The selection of an appropriate expression system is key to producing highly efficient biologics. GOX has been used extensively in industry since the 1950s. The main strains of industrial production GOX are icillium ,76, and icillium . HoweverPichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia lipolytica, and Kluyveromyces lactis [Saccharomyces cerevisiae [Pichia pastoris [Saccharomyces cerevisiae is an earlier microorganism that has been recognized and utilized by humans, but the commercial process of Saccharomyces cerevisiae was limited by the glycosylation of proteins, low protein yield, and plasmid instability [Saccharomyces cerevisiae [Pichia pastoris has attracted attention as an excellent expression host due to its powerful methanol-regulated alcohol oxidase promoter (PAOX1), easy genetic manipulation, highly efficient secretion mechanism, and the ability to perform higher eukaryotic protein modifications [Pichia pastoris expression system, the purification of recombinant proteins is easily conducted due to its limited yield of endogenous secreted proteins. Pichia pastoris can grow to very high cell densities with minimal media, and maintain the genetic stability of recombinant elements during continuous and large-scale fermentation [Pichia pastoris GS115 [Pichia pastoris X33 [Aspergillums niger ATCC 9029 resulted in GOX with a slightly different molecular weight and characterization  with inexpensive components [E. coli by a simple and rapid transformation method. However, the use of E. coli as a heterologous expression host still has some limitations. First, protein expression in the E. coli system is prone to protein non-expression and low soluble protein expression. In addition, the culture of E. coli can form inactive inclusion bodies caused by disulfide bonding, fast production rates, and interactions with chaperones [E. coli. The GOX gene derived from Penicillium amagasakiense was transferred into E. coli for expression, resulting in the formation of inclusion bodies and inactive GOX. Although active enzyme was obtained after denaturation and renaturation, the enzyme was lost [teria/mL . Second,mponents . Third, aperones . Furtherwas lost . Inclusiwas lost .E. coli expression system. To improve the expression of target genes in E. coli, several different strategies have been proposed, including a change in vector, culture conditions, and the gene sequence, as well as the co-expression of molecular chaperones [E. coli at 6–10 °C has been reported as an effective way to overcome the problem of the insolubility of recombinant proteins (mannanase and cellulase) [E. coli, which are normally insoluble [E. coli expression, such as maltose-binding protein (MBP), glutathione-S-transferase (GST), thioredoxin (Trx), and small ubiquitin-modifier (SUMO) [E. coli can become a reality.Notably, GOX always presented as an inclusion body in the aperones . The incllulase) . Moleculnsoluble . Recombinsoluble . Therefor (SUMO) . By takiCell-surface display technology is a useful recombinant technique for expressing functional proteins on the surface of microorganisms, which is achieved through the co-translational fusion of an anchor protein with a functional protein so that the latter is eventually secreted and anchored on the microbial surface ,100. GOXCell-free protein synthesis uses crude cell extracts rather than intact cells to initiate protein synthesis . While iCitrobacter freundii and Heliothis viriplaca, with low similarities in their amino-acid sequences compared with GOX from Aspergillus niger. We also summarize the GOX inhibitors in terms of how they impact GOX activity. We mainly describe strategies for improving production. Although there has been more than 90 years of research on GOX, Aspergillus and Penicillium are the main microorganisms used in the industrial production of GOX, and many questions remain unresolved. First, it is worth finding new strains through selection in nature or metabolic engineering that can produce GOX at a low cost and with high yield, with high biocatalytic activity and stability. Another remaining challenge is understanding the structural properties of GOX, which is a major constraint on their large-scale application. With technological advances, it is believed that GOX will have greater commercial applications in the future.In this paper, we discuss the predictive structures of GOX from


G93A (SOD1G93A) transgenic primary MNs. We found that co-culture of hMSCs and MNs resulted in slightly higher MN numbers, but did not protect against staurosporine (STS)-induced toxicity, implying marginal direct trophic effects of hMSCs. Aiming to elucidate the crosstalk between hMSCs and MNs in vitro, we found high levels of vascular endothelial growth factor (VEGF) and C-X3-C motif chemokine 1 (CX3CL1) in the hMSC secretome. Co-culture of hMSCs and MNs resulted in altered gene expression of growth factors and cytokines/chemokines in both MNs and hMSCs. hMSCs showed upregulation of CX3CL1 and its receptor CX3CR1 and downregulation of interleukin-1 β (IL1β) and interleukin-8 (IL8) when co-cultured with SOD1G93A MNs. MNs, on the other hand, showed upregulation of growth factors as well as CX3CR1 upon hMSC co-culture. Our results indicate that hMSCs only provide moderate trophic support to MNs by growth factor gene regulation and may mediate anti-inflammatory responses through the CX3CL1/CX3CR1 axis, but also increase expression of pro-inflammatory cytokines, which limits their therapeutic potential.Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron (MN) disease characterized by progressive MN loss and muscular atrophy resulting in rapidly progressive paralysis and respiratory failure. Human mesenchymal stem/stromal cell (hMSC)-based therapy has been suggested to prolong MN survival via secretion of growth factors and modulation of cytokines/chemokines. We investigated the effects of hMSCs and a hMSC-conditioned medium (CM) on Cu/Zn superoxidase dismutase 1 Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease affecting motor neurons (MNs) in the primary motor cortex, brain stem, and spinal cord. Patients suffer from rapidly progressive muscle weakness and atrophy, which ultimately lead to death due to respiratory failure within 2–5 years from symptom onset . Of all ALS pathomechanisms include glutamate-mediated excitotoxicity, mitochondrial dysfunction, altered RNA metabolism, disturbed axonal transport, inflammation, oxidative stress, as well as a non-cell autonomous MN death, which is induced by glial cells including astrocytes, microglia, and oligodendrocytes . Due to Although direct cell replacement seems feasible in some neurological diseases such as Parkinson’s disease, in others, such as ALS, the focus of cell therapeutic approaches is rather on generating a protective environment for degenerating MNs. Human mesenchymal stem/stromal cells (hMSCs) are important therapeutic candidates for a variety of diseases, as they promote regeneration and tissue repair due to their growth-factor rich secretome .Moreover, hMSCs can be isolated from almost all tissues and effectively expanded in vitro . AccordiG93A mouse model of ALS. Apart from characterizing CX3CL1/CX3CR1 in the crosstalk of MNs and hMSCs, we tried to elucidate whether changes in this axis coincided with altered gene expression of growth factors and cytokines/chemokines in MNs and/or hMSCs or only arose during co-culture of both cell types. We also aimed to characterize the hMSC secretome and potential pro- or anti-inflammatory changes in transgenic MNs cultured in the presence of hMSC-CM or hMSCs.More important to their therapeutic potential, however, are their low immunogenicity and immunomodulatory effects . hMSCs aAll animal experiments were conducted in strict accordance with the German Animal Welfare Law and were by the Lower Saxony State Office for Consumer Protection and Food Safety .G93A) and wildtype mice were housed under controlled conditions with a light-dark cycle of 12:12 h in groups of a maximum of 6 animals . Animals had free access to food and water at all times. Mice overexpressing the human SOD1G93A gene in a high copy number were purchased from Jackson Laboratories . Hemizygous transgenic male SOD1G93A mice were mated with wildtype C57BL/6 females that were either purchased from Jackson Laboratories  or obtained by own breeding.All transgenic (SOD1n = 5). The production and use of hMSCs has been approved by the Ethics Committee of Hannover Medical School, Germany .Human MSC isolation was performed after written consent from the bone marrow of healthy donors  and enriched with 1% Penicillin/Streptomycin, heparin 2 IU/mL, and 8% human plate lysate in 175 cm2 tissue culture flasks . After four days, cells were washed thrice with 30 mL PBS to avoid blood residues. The medium was changed every 4–5 days and adherent cell growth was microscopically controlled. Cells were subcultured until they reached 80% of confluence and passaged at a density of 2 × 103 cells/cm2. The remaining cells were cryopreserved at a density of 1 × 106 cells/mL in MSC medium with 5% dimethylsulfoxide  and frozen at 70 °C. Cells were used for culture at a low passage (≤4) [The puncture was performed after local anesthesia with a bone marrow puncture needle 15 G  at the pelvic crest. Approximately 10–15 mL of bone marrow was aspirated. Human bone marrow was diluted with a ratio of 1:4 with sterile phosphate-buffered saline (PBS) and the cell count was determined. Cells were cultivated with a concentration of 1–2 × 10age (≤4) ,24.According to the recommendation of the International Society for Cellular Therapy, expression of hMSC surface markers was confirmed by single platform fluorescence-activated cell sorting (FACS) and its capacity to differentiate into chondrocytes, osteoblasts, and adipocytes was analyzed and validated in multiple independent experiments. In order to meet the criteria, collected cells were analyzed for their expression of CD73, CD90, and CD105, as well as the absence of CD3, CD14, CD19, CD34, CD45, and HLA-DR expression in multiple independent experiments .For identification of MSCs, as well as the determination of HLA-DR+ and CD45 expression, the fluorochrome-conjugated monoclonal antibodies (mAb) CD90FITC, HLA-DR PB, CD45PC7  and CD73PE, CD105APC  were used. For cell composition, the fluorochrome-conjugated monoclonal antibodies (mAb) CD90FITC, CD34PE, CD19PC7, CD3APC, CD14 PB, and CD45KO  were used. After 15 min of incubation at ambient temperature in the dark, erythrocyte lysis was carried out by adding NH4Cl solution if necessary. Single platform analysis using flow-count fluorospheres on a Navios™ 10 color flow cytometer  was performed. To distinguish between viable and dead cells, 7-Amino-Actinomycin-D (7-AAD) was used.The average percentage of cells expressing CD73, CD90, and CD105 in the population of bone marrow hMSCs was 97.5% , enriched with 1% Penicillin/Streptomycin. A medium change was performed every 3–4 days. After four weeks cells were washed once with 1xPBS and fixed with 8% PFA. Afterward, cells were washed trice with distilled water and stained with Alizarin Red solution  to detect calcium-rich deposits and SIGMA FAST BCIP/NBT  to identify alkaline phosphatase activity.For adipocyte differentiation, the MSC medium was replaced with the StemPro Adipocyte Differentiation Basal medium  and enriched with 1% Penicillin/Streptomycin for 14 days. After two weeks, cells were washed twice with 1× PBS and fixed with 4% PFA. Cells were stained with Oil Red O  to enable detection of lipid vacuoles.5 cells/well in 6-well plates . After rinsing with 1xPSB, the Neurobasal MN culture medium  containing 5% horse serum , 2% B27 supplement , and 10% Glutamax (Invitrogen) was added. After 24 h, the supernatant was retrieved, centrifuged at 2000× g rpm for 5 min, and rinsed through a 0.22 µm filter to remove cell debris and remaining cells. The obtained conditioned medium was stored at −70 °C until further use.hMSCs were cultured as described above and seeded at a density of 1 × 10G93A males. Embryos (day 12.5) were euthanized as well and the lumbar spinal cords of the embryos were dissected. Enrichment of MN was conducted, as previously described [NTR . Eight-well chamber slides  or 6-well plates  were precoated with poly-L-ornithin , 1:1000 in borate buffer  for 30 min at 37 °C. The diluted poly-L-ornithin was removed and slides/wells were washed twice with distilled water. After washing, slides/wells were coated with laminin , diluted 1:100 in Hanks Balanced Salt Solution  at room temperature (RT) until cell harvest. For cultivation, Neurobasal motor neuron culture medium  containing 5% horse serum , 2% B27 supplement (Invitrogen), and 10% Glutamax (Invitrogen) was used. The horse serum used in the neurobasal medium was previously heat inactivated for 45 min at 56 °C. MN were either plated in neurobasal hMSC-conditioned medium or in an equal amount of hMSCs, dependent on experimental design.Primary MN were obtained by euthanizing pregnant wildtype female animals that were mated with SOD1escribed , by immuG93A) of each experiment. Means were then calculated out of the three independent experiments (according to their genotype) and expressed as means with SEM per genotype.For quantification of MN numbers, three independent experiments were conducted at different timepoints. Two technical replicates per genotype were seeded on two distinct wells. Means were calculated out of the technical replicates and according to the biological replicates (wildtype or SOD1STS is a non-selective protein kinase inhibitor that induces apoptosis in various cell types. After 5 days in vitro (DIV), cells were incubated for 24 h with different concentrations of STS  , dissolved in 1 mM DMSO to test for concentration-dependent neurotoxic effects by immunocytochemistry and LDH assay. The concentration of 0.5 µM was the first concentration showing significant reductions in MN number after 24 h. Further, increasing STS concentrations enhanced this effect; thus, 0.5 μM of STS was used for the main experiments. As a control, cells not exposed to STS were used.LDH is a soluble cytosolic enzyme present in most eukaryotic cells and is released into the culture medium due to damage to the plasma membrane in the case of cell death. The increase in the LDH activity in culture supernatant is proportional to the number of lysed cells. In our experiment, the LDH concentration was measured using the Pierce LDH assay kit, according to manufacturer’s instructions , after 5 DIV and after 24 h of STS exposure. Seventy microliters of supernatant were collected und centrifuged to avoid cell debris, 50 µL of which were incubated at RT with 50 µL of reaction solution. Stop solution was added after a 30 min incubation period and LDH levels were determined at 492/630 nm.On the 5th DIV, cells were incubated for 1 h with the MitoTracker stain (MitoTracker™ Red CMXRos) mixed with neurobasal medium 1:100 nM und washed once with 1× PBS. Cells were fixed in 4% paraformaldehyde for 20 min at RT. Afterward, wells were washed thrice with 1× PBS. Cells were blocked in blocking buffer consisting of 0.2% Triton-X-100 (Sigma), 10% goat serum (Invitrogen), and 2.5% BSA at RT for 30 min. After blocking, primary antibodies were added  in blocking solution and incubated overnight at 4 °C. The next day, wells were washed thrice with 1× PBS. Secondary antibodies  were diluted in blocking buffer and incubated for 2 h at RT. Cells were washed thrice with 1× PBS and once with distilled water. After washing was completed, cells were mounted in mounting medium  containing 0.1% DAPI (Sigma), and visualization took place through fluorescence microscopy . Image acquisition was performed with Cell F and CellSens Dimensions Ink. Software 1.18 , using a 20× objective and a numerical aperture of 0.17.5 cells/well on 6-well plates  in CellGro® MSC medium  enriched with 1% Penicillin/Streptomycin, heparin 2 IU/mL, and 8% human plate lysate. After 5 min (indicated as timepoint 0), 24 and 48 h cell supernatant was collected. As a control, pure MSC medium was used. Media were measured using multiplex technology  ). Pure MSC medium  was used as a negative control, and thus subtracted from all measured values. Supernatants (25 µL) were co-incubated with 25 µL of the missed beads, and 25 µL assay buffer for 45 min at RT in the dark on a plate shaker. After three washing steps, 25 µL mixed biotinylated secondary antibodies were added and incubated for 45 min, followed by three washing steps. Quantification was performed by adding SA-PE (1:100 dilution in assay buffer) for 30 min in the dark. Individual standard curves and concentrations for each analyte were calculated by the Bio-Plex Manager 6.2 software ; the detection range for each analyte was between 1 pg/mL as the lowest and 10 µg/mL as the highest concentrations. Pure MSC medium was used as a negative control, and thus subtracted from all measured values.hMSCs obtained from 5 healthy donors were cultured with a density of 1 × 10−ΔΔCt) [MN in mono-culture and co-cultured with hMSCs were lysed after 5 DIV in order to perform gene expression analysis by qRT-PCR. The medium was removed, cells were washed with 1xPBS, and cells were lysed using the Rneasy Micro Kit , according to manufacturer’s instructions. A total quantity of 1 and 0.2 ng cellular RNA was reverse transcribed to complementary DNA (cDNA) with the QuantiTect Reverse transcription kit , according to the manufacturer’s protocol. qRT-PCR experiments were performed using cDNA from 5, 20, or 50 ng of total RNA from at least four independent experiments. An amount of 1.75 µM forward/reverse primer and Power SYBR-Green PCR Master Mix  or appropriate Assay Mix , combined with TaqMan Universal Master Mix (2×) , were applied. Standard cycling conditions were applied: 90 °C/10 min. and 40 cycles at 95 °C/15 s and 60 °C/1 min. StepOne instrument and software  were used (threshold was set at 0.2). For quantitative analysis, the following reference genes: hypoxanthine phosphoribosyltransferase 1 (HPRT1), peptidylprolyl isomerase A (Ppia), glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH), β-actin and tubulin, and β2-mikroglobulin (b2m), respectively, were applied and expression patterns were analyzed by comparative Ct method (2−ΔΔCt) .For qRT-PCR, specific human and mouse primers were used for analysis of gene expression of hMSCs and mouse primary MNs, respectively. Human- and murine-specific primer pairs were tested with corresponding human and mouse cDNA to confirm the correct length of PCR products via gel electrophoresis on 4% agarose gels. PCR reactions with human primer pairs and murine cDNA or murine primer pairs and human cDNA did not yield PCR products as assessed by gel electrophoresis, thereby confirming species-specificity. Primers are listed in t-test was used, and for the comparison of three or more groups, a one-way ANOVA and Tukey’s multiple comparisons post-hoc test was applied. For two groups with more than two parameters, a two-way ANOVA and Tukey’s multiple comparisons post-hoc test was used. Differences were considered significant in cases when the p-value < 0.05 and is represented as follows: *: p-value < 0.05, **: p-value < 0.01, and ***: p-value < 0.001.Data were expressed as means ± SEM standard error of the mean of at least three independent pooled experiments. Statistical analysis was performed using GraphPad Prism version 9 . For the comparison of two groups, an unpaired G93A mice in vivo [G93A and wildtype MNs were either cultured in monoculture (MN monoculture), co-cultured on hMSCs (MN-hMSC co-cultures), or incubated in hMSC for 5 DIV. MN number was determined by the early MN marker islet1 combined with a marker for mitochondrial activity (MitoTracker). Double positive MNs were counted. Exemplary stainings for all conditions  can be seen in We previously showed that murine MSC-CM exerted neuroprotective effects on MNs due to the release of growth factors and cytokines/chemokines of hMSCs in vitro  and that in vivo . To furt+-islet1+-MNs when MNs were co-cultured with hMSCs (MNs-hMSCs) compared with MN monocultures and MNs incubated in hMSC-CM. This increase was similar for both wildtype and SOD1G93A MNs (There was a significant increase in MitoTrackerG93A MNs .+-MitoTracker+ MNs were determined. Shorter time points (1 h) did not induce MN toxicity and longer ones (48 h) resulted in total MN apoptosis (data not shown). The first significant reduction in MN number was achieved after 24 h, with a concentration of 0.5 µM STS for both wildtype and SOD1G93A MNs when co-cultured on hMSCs. Higher STS concentrations resulted in significantly higher toxic effects. Results were also confirmed by LDH assay, as the first significant increase in LDH levels was detected at a concentration of 0.5 µM STS after 24 h of STS exposure  when exposed to 0.5 μM STS; however, the level of significance was not reached. Wildtype and SOD1G93A MN numbers were slightly increased by hMSC-CM or hMSC co-culture, but no significant neuroprotective effects against STS-neurotoxicity were detected  Modification of MN gene expression of neuroprotective growth factors  in both wildtype and SOD1G93A MNs, and specific upregulation of GDNF only in SOD1G93A MNs, as well as upregulation of the anti-inflammatory chemokine receptor CX3CR1 in both wildtype and SOD1G93A MNs by hMSC co-culture; (e) Upregulation of gene expression of the pro-inflammatory cytokines TNFα, IL6, and IL8 in MNs (with higher levels in SOD1G93A MNs) by hMSC co-culture.The main findings of the present study include: (a) A higher viability of MNs in co-culture with hMSCs, but no significant neuroprotection against STS toxicity; (b) Detection of VEGF, BDNF, CNTF, FGF-2, PDGF, and CX3CL1 in the secretome of hMSCs; (c) Upregulation of anti-inflammatory chemokines (CX3CL1 and its receptor CX3CR1) and downregulation of pro-inflammatory cytokines (IL1b and IL8) in hMSCs when co-cultured with SOD1G93A MNs was not significantly higher compared to wildtype MNs, suggesting that longer culture periods may be necessary to mimic ALS onset in this early in vitro model.Slightly higher MN viability was registered when MNs were co-cultured on hMSCs. Exposure to STS reduced MN numbers in all conditions, but no significant neuroprotective effect of hMSCs against STS-toxicity could be detected. Vulnerability of SOD1G93A mouse model have shown neuroprotective effects of hMSCs [Several in vivo studies of the SOD1of hMSCs ,32,33, bof hMSCs ,35,36,37G93A MNs). Examination of the secretome of hMSCs revealed secretion of BDNF, CNTF, and PDGF-AB, decreasing over time and sustained detection of VEGF, whereas levels of CX3CL1 showed a non-sustained increase. Gene expression of both CX3CL1 and its receptor CX3CR1 were significantly upregulated in hMSCs when co-cultured with SOD1G93A MNs. Contrarily, in MNs co-cultured with hMSCs, CX3CL1 was significantly downregulated, whereas its receptor was significantly upregulated. Interestingly, this effect was not SOD1G93A-dependent. Upregulation of CX3CR1 in MNs in hMSC co-culture could be interpreted as a reaction to the increased secretion and gene expression of CX3CL1 by hMSCs.To elucidate the controversial findings between in vivo studies in mouse models and clinical trials, studies characterizing hMSCs demonstrated that the beneficial effects of hMSCs were mostly exerted by their secretome ,39. ThusCX3CL1  is constitutively expressed in neuronal cells and exerts multiple functions via its CX3CR1 receptor, which is primarily located on microglia, as well as in monocytes, dendritic cells, natural killer cells, and T cells ,41. CX3CG93A mouse model, neuroinflammation is already observed at early stages and becomes more pronounced with greater disease severity and progression [G93A MNs as opposed to co-cultured with wildtype MNs, implying immunomodulatory cross-talk. On the contrary, IL8, together with IL6 and TNFα, were significantly upregulated in SOD1G93A MNs co-cultured with hMSCs compared with wildtype MNs co-cultured with hMSCs, suggesting that hMSCs do not alleviate ALS-related inflammatory mechanisms within this experimental context.Neuroinflammation is one of the main pathomechanisms in ALS . It refegression ,50. Durigression . IL1b, Igression ,53. In lgression ,55. WithG93A MNs co-cultured with hMSCs, GDNF showed a specific upregulation only in SOD1G93A MNs when co-cultured with hMSCs. All of these are well-characterized trophic factors for the central nervous system [G93A genotype, the observed increase in the presence of hMSCs does not seem to reflect ALS-specific regulations. In contrast, GDNF, which was only upregulated in SOD1G93A, implies an ALS genotype-dependent gene regulation associated to the presence of hMSCs.hMSCs secretome analysis revealed a sustained increase in VEGF, a growth factor best known for its angiogenic properties whose protective effects on degenerating MNs have been shown in vitro and in vivo ,57,58. Gs system . Growth s system ,61. As tMultiple studies have shown beneficial effects of growth factors on MNs and in ALS; however, outcomes of clinical trials using growth factors have only marginally prolonged life expectancy of patients so far . AlthougG93A ALS motor neurons, which may limit their therapeutic potential.To conclude, our in vitro analyses provide evidence that hMSCs can provide a beneficial environment for MNs mainly via growth factor secretion and stimulation of growth factor gene expression. These cells modulate MN immune responsiveness and possibly microglial activation through CX3CL1/CX3CR1, but also seem to increase gene expression of pro-inflammatory cytokines in SOD1(a)G93A MNs in co-culture with hMSCs, but no significant protection against STS-induced apoptosis;Higher viability of both wildtype and SOD1(b)G93A MN-hMSC co-culture;Regulation of the inflammatory CX3CL1/CX3CR1 axis in SOD1(c)G93A MNs co-cultured with hMSCs;Increased gene expression of GDNF and pro-inflammatory cytokines IL6, IL8, and TNFα in SOD1(d)Potential limitations of the therapeutic efficacy of hMSCs in ALS due to cellular crosstalk resulting in increased pro-inflammatory rather than anti-inflammatory signaling.


Overall, relapse-free survival tended to be better in patients with low nuclear/high membranous β-catenin expression (n = 9) than in other individuals (n = 51) . Activation of the Wnt/β-catenin signal pathway represented by nuclear β-catenin accumulation was significantly associated with a poor response to NACRT in patients with rectal cancer. Analysis of nuclear β-catenin accumulation before starting treatment might help predict the therapeutic response to NACRT.Although a therapeutic response to neoadjuvant chemoradiotherapy (NACRT) is important to improve oncological outcomes after surgery in patients with locally advanced rectal cancer, there is no reliable predictor for this. The Wnt/β-catenin signal is known to be crucial for the tumorigenesis of colorectal cancer. This study aimed to investigate the association of Wnt/β-catenin signal activation with a pathological response to NACRT. The immunohistochemical expression of nuclear and membranous β-catenin was analyzed in biopsy samples obtained from 60 patients with locally advanced rectal cancer who received curative surgery following NACRT. The association of Wnt/β-catenin signal activation with their clinical outcomes was investigated. Notably, the body mass index of these patients was significantly higher in the low nuclear β-catenin expression group. Moreover, patients in the high nuclear β-catenin expression group tended to have more advanced disease and a higher rate of positive vascular invasion than those in the low expression group. Furthermore, the rate of good histological responses was significantly higher in the low nuclear β-catenin expression group (72% vs. 37.1%,  Neoadjuvant chemoradiotherapy (NACRT) followed by total mesorectal excision (TME) is the standard treatment for locally advanced rectal cancer. It has been reported that NACRT could improve locoregional control with local recurrence rates of approximately 5–9% ,2,3. HowWnt/β-catenin signaling is known to play a crucial role in the regulation of cell proliferation, differentiation, and morphogenesis throughout the body ,11,12. RIn the present study, the association of Wnt/β-catenin signal activation with a pathological response to NACRT in patients with locally advanced low rectal cancer was investigated. In addition, we investigated its association with long-term outcomes in such patients.Overall, 70 patients with locally advanced rectal cancer who received curative surgery following NACRT at Kobe University Hospital from January 2005 to July 2020 were retrospectively analyzed in this study. Inclusion criteria for this study were as follows: histologically proven adenocarcinoma, lower tumor margin below the peritoneal reflection, and cT3/4 or cN+ disease without distant metastasis. In contrast, patients whose biopsy specimens before NACRT were not available for immunohistochemical examination were excluded. Finally, 60 patients were subjected to the final analysis. Tumors were classified according to the tumor-node-metastasis system by the American Joint Committee on Cancer [The Institutional Review Board and Ethics Committee of the Kobe University Graduate School of Medicine (IRB reference number: B210041) provided their approval for this study to be conducted.2/day and leucovorin 75 mg/body/day or capecitabine 1650 mg/m2/day were orally administered for 25 days. Concomitant chemotherapy was initiated on the first day of radiotherapy. The lateral pelvic area was included in the radiation target volume. Surgery according to the TME principle was performed 6–8 weeks after the completion of NACRT. The lateral pelvic lymph node dissection was performed only in patients with clinically positive lateral pelvic lymph nodes based on the pretreatment images, regardless of the clinical response to NACRT. Clinically positive metastasis of the lateral pelvic lymph node was diagnosed by a short axis diameter of ≥7 mm on computed tomography or magnetic resonance imaging scans and/or a high-intensity spot on a positron emission tomography scan. Patients found to have developed distant metastases on post-NACRT imaging studies were excluded from the group of those indicated for curative surgery.Patients with locally advanced low rectal cancer received NACRT comprising a total radiation dose of 45–50 Gy in 25 fractions for 5 weeks as well as an oral 5-fluorouracil-based chemotherapy (tegafur-uracil/leucovorin or capecitabine), as described previously ,21. 45 GThe pathological tumor response to NACRT was determined based on the grading scale according to the Japanese Society for Cancer of the Colon and Rectum guidelines . BrieflyPostoperatively, adjuvant chemotherapy was considered for all patients, regardless of the pathological stage, and follow-up was performed every 3 months for the first 3 years and every 6 months thereafter, as previously reported.Biopsy samples collected before NACRT were fixed in formalin and embedded in paraffin for immunohistochemistry. Serial cross-sections were cut and stained with hematoxylin and eosin to identify the most representative part of the tumor. β-catenin immunostaining was performed on formalin-fixed paraffin-embedded tissue according to the manufacturer’s instructions. Further, the β-catenin-specific monoclonal antibody  was used to detect β-catenin.The expression of β-catenin in the nucleus and membrane of the tumor cells was evaluated by a surgeon working in the pathological department and reviewed by a pathologist without knowledge of the clinical information. The expression of β-catenin in the nucleus or membrane was semi-quantitatively evaluated by calculating the ratio of the number of tumor cells that expressed nucleus or membranous β-catenin to the total number of tumor cells in the tissue section. Further, the ratio was scored as follows: 0 (<1% of positive cells), 1+ (1%–5% of positive cells), 2+ (5%–30% of positive cells), or 3+ (>30% of positive cells). According to the previous studies, the reference value of the ratio considered to be high nuclear β-catenin expression ranges from > 0 % to 30 % and has not been standardized ,19,23,24p-value < 0.1 in the univariate analysis were further subjected to the multivariate analysis. A p-value of <0.05 was considered statistically significant. All statistical analyses were performed with EZR 1.54 , which is a graphical user interface for R 4.2.2 .The chi-square or Fisher’s exact test, as appropriate, was used to perform the comparison of categorical variables. Nonparametric variables were presented as median values and ranges. The Mann–Whitney U test was used to compare these variables. The Kaplan–Meier method was used to perform survival analysis, and the log-rank test was used to perform the univariate survival comparison. Univariate and multivariate analysis was performed to evaluate the predictive factor for relapse-free survival. Variables with a n = 25) and high (n = 35) nuclear β-catenin expression groups. The low expression group included 3 and 22 patients with 0 and 1+ scores, respectively, whereas the high expression group included 18 and 17 patients with 2+ and 3+ scores, respectively. Patient and tumor characteristics are summarized in According to the nuclear β-catenin expression score, patients were classified into the low (Operative outcomes are presented in p < 0.01).Regarding the pathological outcomes, patients in the high nuclear β-catenin expression group tended to have more advanced disease and a higher rate of positive vascular invasion than those in the low expression group . Furtherp = 0.267) or RFS . Regarding comparison between patients with low nuclear β-catenin/high membranous β-catenin expression (n = 9) and others (n = 51), OS and RFS tended to be better in the former , although the differences were not statistically significant  and relapse-free survival (RFS) of each group are shown in nificant .NACRT followed by surgery remains a key treatment for patients with locally advanced rectal cancer, although recent advances in total neoadjuvant therapy appear promising ,26,27,28In the present study, high nuclear β-catenin expression was significantly associated with poor response to NACRT. However, its mechanism is still unclear. Takahashi et al. reported that nuclear β-catenin accumulation contributed to resistance to NACRT possibly through its regulation of cancer stem cells (CSC)/epithelial–mesenchymal transition (EMT) properties . TherefoThe present study could not demonstrate a significant association between the level of nuclear β-catenin expression and RFS or OS. However, importantly, better oncological outcomes were observed in the patients with low nuclear β-catenin/high membranous β-catenin levels. Recurrence developed only in one of those nine patients, and all of them were alive within a median follow-up period of 7 years. These results suggested that the localization and expression patterns of β-catenin have prognostic importance. β-catenin has been reported to be found in four distinct subcellular locations: the plasma membrane, cytoplasm, nucleus, and centrosomes . SeveralThis study’s data imply the two possible roles of Wnt/β-catenin signal for patients with rectal cancer undergoing NACRT. First, evaluation of β-catenin expression using biopsy samples might enable the selection of good or poor responders before treatment initiation. Second, the utilization of the Wnt/β-catenin inhibitor might increase sensitivity to NACRT and improve the response rate. Recently, Leung et al. demonstrated that sulfasalazine—a niclosamide derivative anti-inflammatory drug—could suppress colorectal cancer stemness and metastasis by targeting Kirsten rat sarcoma virus signaling, which was involved in the activation of the Wnt/β-catenin pathway . They prThis study has several limitations. First, it is a retrospective small-scale single-institutional study. Second, the specimen was obtained from biopsy samples, and the invasive front of the tumor was not examined. It was hypothesized that EMT was mainly observed at the invasive front of the tumor , suggestIn conclusion, activation of the Wnt/β-catenin signal pathway represented by nuclear β-catenin accumulation was significantly associated with poor response to NACRT in patients with rectal cancer. Analysis of nuclear β-catenin accumulation before treatment initiation might help predict the therapeutic response to NACRT. Furthermore, the Wnt/β-catenin pathway was considered one possible therapeutic approach for improving the therapeutic response to NACRT.


We found that overexpression of MEG3 facilitated BMP9-induced osteogenic markers, ALP activities, and matrix mineralization. However, knockdown of MEG3 attenuated BMP9-induced osteogenic markers. MEG3 increased the phosphorylation of GSK-3β and the protein level of β-catenin. Pyruvate dehydrogenase kinase 4 (PDK4) can also combine with GSK-3β and increase the latter phosphorylation. Moreover, MEG3 increased the mRNA level of PDK4. The ceRNA analysis showed that MEG3 may regulate the expression of PDK4 via microRNA 532-5p (miR-532-5p). The MEG3-enhanced GSK-3β/β-catenin axis can be attenuated by miR-532-5p, and miR-532-5p inhibitor partly rescued endogenous PDK4 and MEG3-mediated expression of PDK4. MEG3 may potentiate PDK4 and GSK-3β/β-catenin by inhibiting miR-532-5p.Studies reported the positive and negative osteogenic effects of MEG3 in mesenchymal stem cells (MSCs). This study aims at clarifying the osteogenic potential of MEG3 and the underlying mechanism. Bone morphogenetic protein 9- (BMP9-) transfected MSCs were recruited as an osteogenic model  In the nucleus, they regulate gene expression networks by controlling nuclear architecture and transcription; in the cytoplasm, they also modulate mRNA stability, translation, and posttranslational modifications –3. MaterMicroRNAs (miRNAs) are short, noncoding RNAs that function by destabilizing and/or suppressing the translation of their target RNAs . AccumulThe mitochondrial enzyme pyruvate dehydrogenase complex (PDC) irreversibly decarboxylates pyruvate to acetyl coenzyme A (acetyl CoA) and controls the following tricarboxylic acid (TCA) cycle . PDC is β (GSK-3β) is a kind of serine-threonine kinases. As a member of canonical Wnt/β-catenin pathway, the function of GSK-3β is to regulate the translocation of β-catenin into nucleus. When the Wnt receptor Frizzled/LRP is not engaged by ligands, GSK-3β continuously phosphorylate Axin-bound β-catenin at Thr41, Ser37, and Ser33 [β-catenin by E3 ubiquitin ligase β-TrCP [β directly and facilitate the phosphorylation and degradation of GSK-3β [β-catenin is promoted, and free β-catenin level rapidly increases. Finally, the accumulated β-catenin can translocate into nucleus and activate target genes [Glycogen synthase kinase-3nd Ser33 , leadinge β-TrCP . When thf GSK-3β . At the et genes , 26, sucβ/β-catenin axis.In the present study, we confirmed the positive osteogenic effect of MEG3, and elucidated the possible mechanism underlying this effect, which was closely related to the inhibition of miR-532-5p and subsequent activation of PDK4/GSK-3μg/mL), and penicillin (100 U/mL). These dishes, flasks, and plates were cultured in 37°C incubators with 5% CO2. Primary antibodies against Runx2 (sc-390715), OPN (sc-21742), β-catenin (sc-7963), GSK-3β (sc-81462), p-GSK-3β (sc-81496), and PDK4 (sc-518103) were bought from Santa Cruz Biotechnology ; GAPDH (10494-1-AP) was purchased from Proteintech ; COXIV was ordered from Cell Signaling Technology . Dylight 594 conjugated secondary antibody (A23410) was bought from Abbkine (Chinese branch). Alizarin Red S (ST1078) and Alkaline Phosphatase Assay Kit (C3206) were obtained from Beyotime .C3H10T1/2, C2C12, and HEK-293 cell lines were purchased from ATCC . Cells were cultured in dishes, flasks, or plates with Dulbecco's modified Eagle's medium which contained 10% fetal bovine serum (FBS), streptomycin  MEFs were seeded in dishes, respectively. After adherence, the MEFs and OR MEFs were washed twice with PBS and harvested on ice with TRIZOL agent (Invitrogen). The samples were sent to Novogene Biotech Co. Ltd.  for RNA-seq analysis . BrieflyThe ceRNA network was constructed based on the target gene of miRNA and the expression correlation. The TargetScan v7.2, miRanda v3.3a, and RNAhybrid V2.1.2 were used to predict the target genes of miRNA. Given the same miRNA target gene share network, the expression Pearson correlation cutoff (≥ 0.9) for each pair will select ceRNA network edges. The lncRNA-miRNA pairs and miRNA-mRNA pairs were screened to construct the lncRNA-miRNA-mRNA network and visualized by Cytoscape V3.8.2 software.The miR-532-5p mimic, miR-532-5p inhibitor, and their corresponding control were bought from GeneBio . They were transfected with a final concentration of 50 nM into MEFs using Lipofectamine 2000  according to the manufacturer's instructions. The sequence for miR-532-5p mimic, CAUGCCUUGAGUGUAGGACCGU, ACGGUCCUACACUCAAGGCAUG; miR-532-5p mimic NC, UCACAACCUCCUAGAAAGAGUAGA, UCUACUCUUUCUAGGAGGUUGUGA; miR-532-5p inhibitor, ACGGUCCUACACUCAAGGCAUG; miR-532-5p inhibitor NC, UCUACUCUUUCUAGGAGGUUGUGA.The sequences of MEG3-WT, MEG3-MUT, miR-532-5p NC, and miR-532-5p were previously inserted into the vector psiCHECK2, respectively. Then, the recombinant vectors were transfected into HEK-293 T cells with Lipofectamine 2000 according to the group division . The cells from each group were collected after 48 h and subjected to Dual-Luciferase® Reporter Assay System  following the manufacturer's instructions.μL lysis buffer and supernatants for preclearance. The supernatants were incubated with primary anti-PDK4 antibody or IgG overnight (4 degrees Celsius). Next day, the washed beads were added to complexes separately, followed by shaking for 3 h (4 degrees Celsius). Then, these complexes containing beads were collected with the magnetic stand, followed by RIPA buffer wash for 5 times. Finally, the obtained proteins were boiled and subjected to western blot analysis. Primary anti-p-GSK-3β antibody was used for western blot detection.At the designed time point, cells were washed twice with PBS and lysed on ice using RIPA lysis buffer , phosphatase, and protease inhibitors. The cell lysates were then centrifuged (12000 rpm for 10 min), and the supernatants were collected. The magnetic beads of protein G  were washed with 30 OR MEFs were constructed by small RNA library screening and ectopic bone formation. Briefly, MEFs were transfected with retrovirus containing the small RNA library, followed by BSD screening for the positively transfected MEFs. Then, the positively transfected MEFs were transfected with Ad-BMP9 for several rounds, and the living cells were screened out for ectopic bone formation. Subsequently, the ectopic bones were retrieved, and the uncalcified cells were separated for the second round of BMP9 transfection and ectopic bone formation. After the third round of screening, the uncalcified cells were retrieved and regarded as OR MEFs.β-glycerophosphate, and L-ascorbic acid. Sodium β-glycerophosphate and L-ascorbic acid were dissolved in DMEM and filtered using a 0.2-μm filter. Dexamethasone was dissolved in DMSO. The obtained solutions were added to DMEM containing 10% FBS. The final concentration of dexamethasone was 10 nM; sodium β glycerophosphate was 10 mM, and L-ascorbic acid was 50 μg/mL [OIM contains dexamethasone, sodium 50 μg/mL . For ostAt the preset timepoints, total RNA was extracted using TRIZOL reagent , purified, and used for reverse transcription (RT) and cDNA generating. The resultant cDNA was diluted 5-10 folds for qPCR assay. The expression of the housekeeping gene GAPDH was used to normalize the PCR results. MEG3, Runx2, DLX-5, etc. were regarded as the target genes. The primer sequences used in this study were presented in μL lysis buffer on ice. Subsequently, the lysate was transferred into 1.5 mL Eppendorf tubes, followed by the addition of 15 μL β-mercaptoethanol and 75 μL loading buffer in each tube. Then, the tubes were bathed in boiling water for 15 min and stored in -20°C or -80°C. The samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the gels containing target protein were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were bathed in 5% bovine serum albumin (BSA) for 1 h at room temperature and then incubated in the primary antibody solutions at room temperature for 1.5 h or at 4°C overnight. The membranes were washed five times with Tris-buffered saline Tween20 (TBST) and incubated in corresponding secondary antibodies  for 0.5 h at room temperature. Finally, the membranes were washed five times with TBST, and the images of the target bands were taken using SuperSignal West Pico Chemiluminescent Substrate (SWPCS) . The primary antibodies were diluted in 5% BSA with a ratio of 1 : 1000; the secondary antibodies were diluted 1 : 3000 in TBST.At the designed timepoint, the cells in 6-well plates were washed twice with phosphate buffered saline (PBS) and lysed with 300 μL/well) from Beyotime Alkaline Phosphatase Assay Kit for 15 min in the dark. Briefly, the working solution contained BCIP, NTB, and ALP buffer, at a ratio of 33 μL : 66 μL : 10 mL. These plates were dried and scanned for data analysis. For microscopic images, the wells were photographed using a fluorescence microscope . These results were repeated in at least three independent experiments.At the designed timepoint, the cells in 12-well or 24-well plates were washed twice with PBS and stained with the working solution . Finally, each well was bathed in 10% acetic acid, and the absorbance at 405 nm was detected using a microplate reader as previously described , 32. TheAt the designed timepoint, the cells in 48-well plates were washed with PBS and fixed with 4% paraformaldehyde. Subsequently, the cells were bathed in 0.3% TritonX-100, followed by PBS wash for three times. Then, the cells were incubated with goat serum  at room temperature for 1 h and bathed in the primary antibody solutions (1 : 200) at 4°C overnight. The cells were washed three times with PBS the next day, followed by incubation in the dark with Dylight 594 conjugated secondary antibody (1 : 50) at 37°C for 1 h. Then, the cells were washed with PBS for three times and stained with 4,6-diamino-2-phenyl indole (DAPI) for 5 min. The DAPI was removed, followed by another three times of PBS wash. Finally, the cells were bathed with 80% glycerin and used for immunofluorescent image taken under a fluorescent microscope or confocal laser scanning microscope. The dilution rate was 1 : 10000 for DAPI. These results were repeated in at least three independent experiments.6 per injection) at the flanks of athymic nude mice . Four weeks after transplantation, the mice were euthanized, and the ectopic bone tissues were retrieved for μCT assessment and histological analysis. No bone masses were found in Ad-GFP-transfected group. The nude mice were all ordered from Animal Centre of Chongqing Medical University . The animal experiments were approved by the Ethics Committee of Chongqing Medical University.MEFs were transfected with Ad-GFP, Ad-BMP9, Ad-BMP9+Ad-MEG3, and Ad-BMP9+Ad-siMEG3. Twenty-four hours after transfection, cells were collected and resuspended in cold PBS for subcutaneous transplantation  for 4 weeks, dehydrated by ethanol, and embedded with paraffin. Then, the paraffin-covered masses were sliced and subjected to immunofluorescent staining, hematoxylin-eosin (H&E) staining, and Masson's trichrome staining after deparaffinization and rehydration.μCT . The three-dimensional reconstructions and analysis were performed following the software linked to the scanner (μCT 516.1).The harvested samples of bone tissue were scanned with http://rsbweb.nih.gov/ij/download.html) software was used for quantifications. For western blot results, the ratio of target bands to their GAPDH/COXIV was normalized to control, followed by further statistical analysis. For ALP results, the images of each well were measured for further statistical analysis. For histological evaluations, such as IHC, H&E, and Masson's trichrome staining, the positive area percentage was used for quantification and statistical analysis.The NIH ImageJ (t-test. p values less than 0.05 were considered statistically significant, and p values less than 0.01 were considered remarkably significant.The software GraphPad Prism 8 was used to analyze the mean ± SD values and the significant differences between two groups by means of two-tailed Student's in vitro osteogenic model, we found that MEG3 potentiated the level of early osteogenic markers, such as Dlx-5 and RUNX2 (Figures OR MEFs were more difficult to be induced into osteogenic progenitor cells and osteoblasts. The RNA-seq analysis between MEFs and OR MEFs showed that the expression of MEG3 was lower in OR MEFs , and MEG Figures . The sam Figures , western Figures , and mat Figures . These rTo investigate the opposite effect of MEG3 on BMP9-induced osteogenic differentiation, we constructed the recombinant adenovirus for knockdown of MEG3 Figures . The comin vivo. By ectopic bone formation, we found an increased mean volume when BMP9 was combined with MEG3, and a decreased mean volume when BMP9 was combined with siMEG3 (μCT scan (in vivo while siMEG3 attenuated it (in vivo can be promoted by MEG3 and attenuated by knockdown of MEG3.We next explored the effect of MEG3 on BMP9-induced osteogenic differentiation h siMEG3 . The thrμCT scan  and analμCT scan  showed tuated it . The samuated it . These huated it  These daβ-catenin in BMP9-induced ectopic bone tissues (β and the expression of β-catenin in BMP9-induced MEFs (Figures β-catenin into nucleus (β and the expression of β-catenin (Figures β/β-catenin axis.We next sought to explore the mechanism of the positive osteogenic effect of MEG3. IHC staining showed that MEG3 increased the protein level of  tissues . Western Figures . Confoca nucleus . On the  Figures . These rβ and the protein level of β-catenin (Figures β (β/β-catenin axis by promoting the phosphorylation and degradation of GSK-3β.As PDK4 is involved in vascular calcification and osteogenic differentiation, we constructed recombinant adenoviruses for overexpression and knockdown of PDK4 Figures . OIM cul Figures , which w Figures . Furtherigures β . These dβ/β-catenin axis. Therefore, we investigated the relationship between MEG3 and PDK4. qPCR results showed that MEG3 increased the mRNA level of PDK4 with or without the interference of BMP9 (MEG3 and PDK4 showed the same positive effect on GSK-3 of BMP9 . However of BMP9 . Given t of BMP9 , in whic of BMP9 . We seleβ and the protein level of β-catenin (Figures β/β-catenin axis can be negatively regulated by miR-532-5p. The possible mechanism of MEG3 on PDK4 and osteogenesis was demonstrated in Based on the previous results, we further detected the effect of MEG3 on the expression of miR-532-5p. The qPCR results showed that MEG3 inhibited the expression of miR-532-5p while knockdown of MEG3 promoted this expression with or without the participation of BMP9 Figures . Western Figures . However Figures . The dua Figures . Finally Figures . Accordi Figures . These din vitro and in vivo, while knockdown of MEG3 attenuated BMP9-induced osteogenic differentiation. Overexpression of MEG3 and PDK4 both increased the phosphorylation of GSK-3β and the protein level of β-catenin. Moreover, MEG3 promoted the mRNA level of PDK4, indicating that MEG3 may potentiate osteogenesis via PDK4-mediated GSK-3β/β-catenin pathway. The ceRNA network showed that miR-532-5p was involved in the effect of MEG3 on PDK4, which was confirmed by dual luciferase reporter and the recruitment of miRNA mimic and inhibitor. Hence, our study suggests that MEG3 may promote PDK4-mediated osteogenic differentiation of GSK-3β/β-catenin by acting as a ceRNA, sponging miR-532-5p, and offsetting the inhibition of miR-532-5p to PDK4.In this study, we investigated the effect of lncRNA MEG3 on osteogenic differentiation in MSCs and demonstrated the possible mechanism based on the experimental results. We found that MEG3 enhanced the osteogenic potential of BMP9 both As a kind of lncRNA, MEG3 has been widely regarded as a tumor suppressor and studied in cancer research, such as squamous cell carcinoma, colorectal cancer, and glioblastoma , 6, 33. β-catenin signaling pathway plays an established role in osteogenesis and impacts on arterial calcification [β and the protein level of β-catenin (Figures β-catenin more specific, that PDK4 may combine with GSK-3β and potentiate the latter phosphorylation, leading to the accumulation of β-catenin (β, the protein level (Figures β-catenin (β-catenin in liver cancer cells and malignant melanoma cells [It has been reported that PDK4 plays a critical role in vascular calcification , 39. PDKfication –42, we d Figures . The co--catenin . Similar Figures , and the-catenin . Previouma cells , 44. ThelncRNAs often act as ceRNAs to regulate osteogenic differentiation and adipogenic differentiation in MSCs via sponging certain miRNAs , 45, 46.β, and the subsequent accumulation and translocation into nucleus of β-catenin. Although we investigated the expression of β-catenin from whole cell perspective (Figures β-catenin in the cytoplasm and in the nucleus separately, which gains persuasiveness to the effect of PDK4 on β-catenin. Similarly, our co-IP experiments failed to immunoprecipitated GSK-3β and western blotting PDK4 reversely (It is well known that canonical Wnt signaling is based on Wnt-induced phosphorylation, degradation of downstream GSK-3 Figures , we faileversely . Furthereversely , but a lβ/β-catenin axis and osteogenesis via sponging miR-532-5p and the subsequent activation of PDK4.Our study suggests that MEG3 can positively potentiate the osteogenic differentiation in BMP9-induced osteogenic model. MEG3 may chiefly enhance GSK-3


To explore the effect of the Wnt/β-catenin signaling pathway inhibitor FH535 on the proliferation of MKN45 gastric cancer cells. The cell proliferation index (PI) and apoptosis index (AI) were measured by flow cytometry. The expression levels of β-catenin, c-myc, and cleaved caspase-3 in MKN45 gastric cancer cells were detected.  An MTT colorimetric assay was used to detect the inhibitory effect of the Wnt/β-catenin signaling pathway inhibitor FH535, MKN45 gastric cancer cells showed obvious shrinkage, death, and cell density decrease. MTT showed that the A value of MKN45 gastric cancer cells in FH535 group was significantly lower than that in the control group (P < 0.05). The survival rate of MKN45 gastric cancer cells in FH535 group was significantly lower than that in the control group (P < 0.05). The cell cycle of gastric cancer was arrested in G0/G1 phase. The expression levels of β-catenin and c-myc protein in MKN45 gastric cancer cells in FH535 group decreased significantly (P < 0.05), while the expression level of cleaved caspase-3 protein increased significantly (P < 0.05).  After using the Wnt/β-catenin signal molecule can maintain the proliferation of gastric cancer cells. Inhibition of the Wnt/β-catenin signaling pathway can inhibit the proliferation of gastric cancer cells and promote the apoptosis of MKN45 gastric cancer cells. The Wnt/ Cleaved caspase-3 plays an important role in cell apoptosis. Whether it is a mitochondrial pathway or a dead recipient pathway, it will trigger the caspase apoptosis reaction at the end, which will activate cleaved caspase-3 located downstream of the apoptosis reaction and promote cell apoptosis [47-48]. In this study, we compared and analyzed the expression of the Wnt signaling pathway related proteins in gastric cancer cells after using the Wnt/β-catenin signaling pathway inhibitor. In this study, compared with the control group, the expression levels of β-catenin and c-myc protein in MKN45 gastric cancer cells in the FH535 group were significantly decreased (P < 0.05), and the expression level of cleaved caspase-3 protein was significantly increased (P < 0.05). Therefore, the Wnt/β-catenin signaling pathway inhibitor can positively promote the apoptosis of gastric cancer cells.The Wnt signaling pathway is ubiquitous in all kinds of animals and plays an important role in regulating the normal development of embryos and participating in cell proliferation and differentiation. Wnt signaling pathway mainly includes classical the Wnt signaling pathway, cell polarity regulation pathway, and Wnt/Ca2+ pathway. At present, research mainly focuses on the classical Wnt signaling pathway. Although the Wnt signaling pathway is composed of various proteins, the changes of various regulatory factors are mainly realized by changing the concentration of  from it . C-myc ior cells . Studiesmyc gene . The groβ-catenin signaling pathway can inhibit the proliferation of gastric cancer cells and accelerate their apoptosis. Its mechanism may be related to the Wnt/β-catenin signaling pathway to regulate the expression level of downstream targets such as β-catenin, c-myc, and caspase-3, thus regulating the cell cycle and apoptosis of gastric cancer cells.To sum up, inhibiting the Wnt/