SGO1 downregulation on tumor growth and metastasis in TBNC. To that end, female NOD-SCID Gamma (NSG) mice were injected with 2.5 × 106 shRNA Control (n = 10) or shRNA SGO1 (n = 10) MDA-MB-231 cells. After eight weeks, the number of mice with metastasis to the lymph nodes was calculated. Primary and metastatic tumors, as well as lung and liver tissue, were harvested, measured, sectioned, and stained with hematoxylin and eosin (H&E) stain.Triple-negative breast cancer (TBNC) is an aggressive breast cancer subtype with a poor prognosis. Shugoshin-1 (SGO1) protects chromatids from early separation. Previous studies from our group have demonstrated that transient SGO1 downregulation suppresses early stages of metastasis  in TNBC cells. Thus, the inhibition of SGO1 activity may represent a potential therapeutic intervention against cancers that progress to metastasis. Therefore, we aimed to investigate the effects of sustained shRNA-mediated SGO1-treated mice group, while metastasis to the liver tends to be lower in cells with downregulated SGO1, but it did not reach statistical significance. Furthermore, sustained SGO1 downregulation significantly reduced cell proliferation, cell migration, and invasion which correlated with lower levels of Snail, Slug, MMP2, MMP3, and MMP9.Tumor growth and metastasis to the lymph nodes and lungs were significantly reduced in the shRNA The supression of SGO1 activity in TNBC harboring dysregulated expression of SGO1 may be a potential target for preventing breast cancer growth and metastasis.The online version contains supplementary material available at 10.1186/s13008-023-00088-5. Breast cancer is the most common cancer diagnosed and the number one cause of cancer-related deaths in women worldwide ]. BreastOur laboratory focuses on the study of TNBC. This subtype represents 15% to 20% of all primary breast cancers and occurs most frequently in African American and Hispanic women at a younger age (< 50 years of age) . Higher A potential therapeutic target for TNBC is Shugoshin-1 (SGO1) or mitotic kinases that interact with SGO1, including Nek2A , Bub1 1, TTK  mice. We explore the effects of downregulation by assessing cell proliferation, viability, apoptosis, and EMT in vitroSGO1 were injected in NSG mice. Our results showed that SGO1 downregulation significantly reduced tumor growth and metastasis of TNBC to the lungs and lymph nodes. In vitro studies using the same cell lines showed that EMT protein expression and cell invasion were associated with the reduction in the levels of EMT transcription factors and MMPs. These results suggest that SGO1 is a potential therapeutic target for TNBC.To test our hypothesis, MDA-MB-231 cells expressing either shRNA Control or shRNA SGO1  or negative scrambled shRNA Control  lentiviral particles. The cells were cultured as described in our previous publications [The MDA-MB-231 (HTB-26) cell line was purchased from American Type Culture Collection  and transduced with shRNA ications , 27.SGO1 were lysed and Western blot protocol was performed according to our published protocols [To investigate the effects of SGO1 downregulation on EMT protein expression, MDA-MB-231 cells expressing shRNA control or an shRNA targeting SGO1 , SNAI1 , SNAI2 , TWIST1 , ZEB1 , MMP2 , MMP3 , MMP9 , CDH1 , CDH2 , VIM , and TJP1  were evaluated. GAPDH  mRNA levels were used as a control. The 2−ΔΔCT method was used to calculate fold changes relative to the control and the average of three independent experiments was reported.To investigate the effects of SGO1 downregulation on EMT mRNA expression, total RNA was extracted using the RNeasy Mini kit  following the manufacturer’s instructions. RNA was quantified and reverse transcribed, and quantitative real-time PCR (qPCR) analysis was performed according to our published protocols . The mRNSGO1 using our published methods [BioCoat Matrigel Invasion Chambers  and BioCoat Control Cell Culture Inserts  were used to evaluate the invasion and migration of MDA-MB-231 cells treated with shRNA Control or shRNA  methods . Images To investigate the effects of SGO1 downregulation on cell proliferation and viability, a Cell Counting Kit-8 (CCK8) analysis  was performed as described previously . The aveSGO1 were seeded at a density of 1 × 106 and further trypsinized and washed with PBS 1 × before proceeding with the Annexin V Apoptosis Detection Kit I  protocol. Both cell lines, expressing shRNA Control, or shRNA SGO1 were prepared as follows for flow cytometry: Unstained, PE Annexin V (no 7-AAD), and 7-AAD (no PE Annexin V). Flow cytometry analysis was performed using the FACSMelody instrument within 1 h of staining. The average from three independent experiments was reported for the analysis.To investigate the effects of SGO1 downregulation in apoptosis, MDA-MB-231 treated with shRNA Control or shRNA NSG females (4 weeks old) were obtained from Jackson Laboratories, Inc. (005557). Mice were allowed to adapt under a sterile and pathogen-free environment for two weeks. The mice had free access to distilled water and food were on a 12 h light–dark cycle, and were housed in groups of five per cage. The mice were observed daily by staff, including a veterinarian. For any surgical procedure we used inhaled anesthesia to minimize pain and discomfort. After surgery, the animals were allowed to recover. The parameters to determine a premature endpoint included failure to feed, mood, or groom, or when the tumor reached 10% of body weight.6 MDA-MB-231 (shRNA Control or shRNA SGO1) cells were injected into both posterior mammary fat pads of 16–18 weeks old female NSG mice according to our published protocols [2 after 8 weeks post-injection or when the mice exhibited severe discomfort and pain evidenced by failure to move, groom, or feed.To investigate the effects of SGO1 downregulation in tumor growth, a total of 2.5 × 10rotocols . To miniTo investigate the effects of SGO1 downregulation in metastasis, the liver, lungs, primary tumors, and secondary tumors were fixed in 10% formalin buffer and embedded in paraffin, as previously described . The tisFor all data analysis, we used the software GraphPad Prism version 8.4.3 (686). The values are represented as mean + /− SEM for n = 3 for all in vitro experiments and n = 10 per group for in vivo experiments. The data were considered significant if the p-value was < 0.05. A Student t-test  was used to compare differences between groups. A two-way ANOVA was used to compare differences in groups with more than one categorical variable. For the metastasis studies, a Fisher exact test was performed. To determine the appropriate sample size for measuring tumor growth, we performed the following power analysis: a sample size of 8 mice per group will have 95% power to detect a mean difference of at least 0.3 (with SD = 0.1) using the Hsu (With Best) multiple comparison test at a two-sided 5% type I error using one-way ANOVA.Previous studies from our laboratory have shown a role for SGO1 in early metastasis  of TNBC cell lines . HoweverSGO1 group although it does not reach statistical significance , Figs. SGO1 group , Fig. Metastasis to the lungs, livers, and lymph nodes was then detected. Metastasis to the lungs is more likely to occur in the shRNA control group, compared to the shRNA SGO1 group , Figs. SGO1 in primary tumors and found significant differences in cells on metaphase and anaphase but not on prometaphase or telophase   can be tOur findings indicate that SGO1 plays a novel role in TNBC by regulating the transcription factors Snail and Slug. These transcription factors in turn modulate EMT genes , cell invasion, migration, and lung metastasis Fig. . TherefoAdditional file 1: Figure S1 a-b. Representative images of primary tumors recovered from NSG mice treated with MDA-MB-231 cells expressing shRNA Control (5-10) or shRNA SGO1 (15-20). Figure S2. Original Western blot films showing SGO1 (a) and β-actin (b) expression in tumor lysates from NSG mice treated with MDA-MB-231 cells expressing shRNA Control (1-4) or shRNA SGO1 (5-8). Figure S3. Original Western blot films showing SGO1 (a), Snail (b), and β-actin (c) expression in MDA-MB-231 cells expressing shRNA Control  or shRNA SGO1  from three independent experiments. Figure S4. Original Western blot films showing Slug (a), Zeb1 (b), and β-actin (c) expression in MDA-MB-231 cells expressing shRNA Control  or shRNA SGO1  from three independent experiments. Figure S5. Original Western blot films showing Tiwst1 (a) and β-actin (b) expression in MDA-MB-231 cells expressing shRNA Control  or shRNA SGO1  from three independent experiments. Figure S6. Original Western blot films showing ZO1 (a) and β-actin (b) expression in MDA-MB-231 cells expressing shRNA Control  or shRNA SGO1  from three independent experiments. Figure S7. Original Western blot films showing E-cadherin and Vimentin (a) and β-actin (b) expression in MDA-MB-231 cells expressing shRNA Control  or shRNA SGO1  from three independent experiments.


GSK3β interacting protein (GSKIP) is a small A-kinase anchor protein previously reported to mediate the N-cadherin/β-catenin pool for differentiation in SH-SY5Y cells through overexpression of GSKIP to present the neuron outgrowth phenotype. To further investigate how GSKIP functions in neurons, CRISPR/Cas9 technology was utilized to knock out GSKIP (GSKIP-KO) in SH-SY5Y. Several GSKIP-KO clones resulted in an aggregation phenotype and reduced cell growth without retinoic acid (RA) treatment. However, neuron outgrowth was still observed in GSKIP-KO clones treated with RA. The GSKIP-KO clones exhibited an aggregation phenotype through suppression of GSK3β/β-catenin pathways and cell cycle progression rather than cell differentiation. Gene set enrichment analysis indicated that GSKIP-KO was related to epithelial mesenchymal transition/mesenchymal epithelial transition (EMT/MET) and Wnt/β-catenin/cadherin signaling pathways, suppressing cell migration and tumorigenesis through the inhibition of Wnt/β-catenin mediated EMT/MET. Conversely, reintroduction of GSKIP into GSKIP-KO clones restored cell migration and tumorigenesis. Notably, phosphor-β-catenin (S675) and β-catenin (S552) but not phosphor-β-catenin (S33/S37/T41) translocated into the nucleus for further gene activation. Collectively, these results suggested that GSKIP may function as an oncogene to form an aggregation phenotype for cell survival in harsh environments through EMT/MET rather than differentiation in the GSKIP-KO of SH-SY5Y cells. Glycogen synthase kinase interaction protein (GSKIP) is the smallest A-kinase anchor protein (AKAP)  GSKIP to inhibit neurite outgrowth  of GSKIP, we utilized CRISPR/Cas technology to knock out GSKIP in SH-SY5Y to investigate whether GSKIP functions in cell differentiation or proliferation. Surprisingly, three GSKIP-KO cell clones resulted in an aggregated phenotype and reduced cell growth relative to GSKIP WT. Bioinformatics analysis tools indicated that GSKIP-KO was related to the epithelial mesenchymal transition/mesenchymal epithelial transition (EMT/MET) phenotype and highly associated with the Wnt/β-catenin/cadherin signaling pathway. Also, EMT was mediated by the critical mechanisms through which GSKIP-KO suppressed cell migration and tumorigenesis of SH-SY5Y cells via inhibition of Wnt/β-catenin. Furthermore, the phosphorylation of β-catenin, notably phosphor-β-catenin (S675) and β-catenin (S552), are involved in the translocation into the nucleus and subsequent downstream gene activation and upregulation of N-cadherin for N-cadherin-mediated β-catenin pools in the context of GSKIPs. Finally, GSKIP may function as an oncogene to aid cell survival in harsh environments through the formation of aggregation phenotypes, rather than differentiation through EMT/MET signaling in the GSKIP-KO of SH-SY5Y human neuroblastoma cells.2. When cells were 40–50% confluent, differentiation was initiated by the addition of 10 µM all-trans-retinoic acid  for 6 days. This treatment was replaced every 2 days to replenish RA in the culture media and the cells were analyzed by phase-contrast light microscopy. Images were captured with NIS-elements software.The human neuroblastoma SH-SY5Y cell line was purchased from the American Type Culture Collection  and cultured in DMEM/F12 medium  supplemented with 10% fetal bovine serum  and 1% streptomycin/penicillin (Gibco) at 37 °C with 5% COhttp://crispr.mit.edu/) provided by the Biotools company . The plasmids were transfected into the cells using TOOLSFect Transfection Reagent (Biotools) according to the manufacturer’s instructions. For the CRISPR/Cas-assisted gene, cells were transfected with the plasmids Cas9-plasmid (pRGEN-Cas9-CMV), surrogate-reporter, and gRNA-plasmid (pRGEN-Human-GSKIP), then 48 h after transfection, the cells were treated with 2 μg/ml of puromycin for three days. Dissociated cells were collected and resuspended in Dmem/F12 medium and then analyzed and sorted with a FACS Aria II . Enrichment of cells with RGEN activity was accomplished by single-cell sorting of HA epitope-tagged positive cells into 96-well plates. The colonies were isolated with the cloning cylinders, and the GSKIP sequences were analyzed with a T7 endonuclease (T7E1) assay report, F-PCR report, DNA sequencing, and western blotting . All experimental procedures were conducted following the manufacturer’s protocol, and mutated nucleotides were verified by DNA sequencing with an ABI PRISM 3730 Genetic Analyzer .To generate GSKIP deficient GSKIP-KO cells, CRISPR/Cas9-mediated gene  editing was performed. Both strands of oligo DNAs encoding for one gRNA that specifically targets the GSKIP exon 2 sequences  and pEGFP-GSKIP plasmid DNA (2 μg) were transfected into the cells using Lipofectamine 3000  according to the manufacturer’s instructions. After 48 h, the cells were cultured in fresh medium and further assayed.For transient transfections, approximately 5 × 10Total RNA was extracted using RNA Isolater Total RNA Extraction Reagent  and. quantified using an Epoch spectrophotometer with a Take3 plate . Then, 1,000 ng of total RNA was reverse transcribed using a HiScript III RT SuperMix for qPCR (+ gDNA wiper)  according to the manufacturer’s instructions. The cDNA was amplified using the TopQ Gene Probe Assay for human GAPDH, GSKIP, and GSKIP-CRISPR . Quantitative PCR was performed with ChamGE Probe qPCR Master Mix  in a 10 µL reaction on the StepOne Plus Real-Time PCR System  as per the manufacturer’s protocol. Then, 10 ng of cDNA was amplified in triplicate with the appropriate non-template controls. Amplification data were normalized to GAPDH expression and quantified using the 2-∆∆Ct method. Quantitative PCR data showed a variability coefficient of Ct always lower than 2% of mean values.Protein was extracted using RIPA lysis buffer  and quantified via the Bio-Rad DC protein assay . The protein samples were mixed with SDS loading buffer and separated on an 8–12% SDS-PAGE gel and transferred to PVDF membrane . The membrane was then blocked with 5% BSA (Sigma) at room temperature for 1 h in Tris-buffered saline-Tween 20 . After washing in TBS-T, membranes were incubated overnight at 4 °C in 2.5% BSA with the appropriate primary antibodies and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. All antibodies and dilutions are shown in Table S2. The immunoblot signals were developed using the Super Signal Ultra chemiluminescent reagent  and detected by a CCD camera .Total RNA was harvested and purified using Trizol® Reagent  and the quality was determined using an Agilent 2100 Bioanalyzer with RNA 6000 labchip kit . Experiments were performed and analyzed as a service at Welgene lnc. The statistical significance of the expression data was determined using fold change. Pathway analyses were performed using Ingenuity Pathway Analysis (IPA) software. Statistical significance of the expression data was determined using z-score and P-value.2. Then, 10 μl of the cell counting Kit-8  solution was added to each well at four or five-time points and incubated for 1 h. The change in absorbance at 450 nm was measured photometrically and the experiment was repeated three times.The stable transfectant pEGFP-vector, pEGFP-GSKIP, vehicle (SH-SY5Y), GSKIP-KO#1, GSKIP-KO#7 and GSKIP-KO#9 cells were plated on 96-well plates in DMEM/F12 medium and incubated at 37 °C with 5% COCell survival was measured by a colony formation assay. Cells were seeded in 6-well plates at a density of 1000 cells per well for pEGFP-vector, pEGFP-GSKIP, vehicle (SH-SY5Y), GSKIP-KO#1, GSKIP-KO#7, and GSKIP-KO#9 cells, and allowed to grow for 10–14 days until visible colonies formed. Colonies were fixed with methyl alcohol and stained with crystal violet before the absorbance was measured at 570 nm to calculate the colony formation rate.5 cells/well) and medium supplemented with Dmem/F12 medium supplemented with 10% FBS was added to the bottom chamber as a chemoattractant. Following 16–18 h incubation at 37 °C, cells that migrated through the membrane (migration) were fixed and stained with 0.1% crystal violet (Sigma-Aldrich). The number of cells was counted in three random fields under 100 × objective lens.Migration assays were conducted in 24-well hanging cell culture inserts . Cells resuspended in 300 μl serum-free medium were added to the top chamber (1 × 105 cells/70μL/well) were seeded into each well of a 24 well with a culture insert . Cell debris was removed by washing with PBS, and the cells were cultured in media. Images were captured after wounding and the distance migrated by the cell monolayer to close the wounded area at 0, 24, and 36 h was measured. The wound closure rate was equal to the recovered distance divided by the original width of the scratch.Cell migration was determined by the wound healing assay using IBIDI Culture Inserts . Cells  could induce cell cycle progression by increasing the accumulation of β-catenin in the nucleus and downregulating the association of β-catenin and N-cadherin in the membrane. GSKIP affects the transcriptional state of the cell in neuron development and neuron outgrowth by regulating the functional interplay of the GSK3β/β-catenin, β-catenin/Cyclin D1, and β-catenin/N-cadherin pools during RA treatment in SH-SY5Y cells. Therefore, we revisited how GSKIP-OE regulated β-catenin and N-cadherin during RA treatment, reintroducing GSKIP into SH-SY5Y cells and verifying the OE status of GSKIP by western blotting  sequence included CGG PAM targeted by gRNA in a CRISPR Cas9 system, as shown in Fig. The three GSKIP-KO cell clones  were used to further investigate the function of GSKIP in neuron outgrowth, demonstrating that cell proliferation was suppressed Fig. A and celBioinformative tools were used to analyze the possible molecular mechanisms and potential genes involved in the cell aggregation phenotype. GSKIP-mRNA interactions were used to identify the common up- and downregulated differentially expressed genes (DEGs) in the vehicle with three different GSKIP-KO SH-SY5Y cell clones  Fig. A. After The status of EMT and MET are involved in the initiation of metastasis, pluripotent stem cell reprogramming, and wound healing Kalluri . ConversGSKIP was reintroduced into the three GSKIP-KO cell clones to determine whether GSKIP modulates the interplay with EMT and MET  but also β-catenin (S675) and β-catenin (S552), which translocate into the nucleus to promote cell proliferation and stabilize the N-cadherin/β-catenin complex (pool) signaling pathways for the EMT phenotype. Conversely, the knockout of GSKIP downregulated phosphor-β-catenin (S675) and β-catenin (S552) and attenuated N-cadherin/β-catenin complex (pool) signaling pathways to present the MET phenotype (cell aggregation). It was also demonstrated that the N-cadherin/β-catenin complex has a major role in the EMT/MET phenotype in tumorigenesis in neuron SH-SY5Y cells. Our study revealed new mechanisms through which GSKIP regulates the EMT/MET phenotype through the GSKIP/GSK3β/β-catenin/N-cadherin axis for new insights into the neuronal function and behavior of SH-SY5Y human neuroblastoma cells.According to our previous study, the overexpression of GSKIP upregulates p-GSK3β (S9) in RA-mediated neuron outgrowth , thereby preventing the nuclear translocation of β-catenin and β-catenin/T-cell factor (TCF)-mediated transactivation and impedes Wnt signaling and the acquisition of mesenchymal traits  is viewed as a phosphorylated form of β-catenin through the suppression of GSK3β activity by GSK3β (S9), followed by ubiquitination and degradation. Unphosphorylated β-catenin in the cytosol migrates to and accumulates in the nucleus to bind TCF to activate downstream factors , our data suggest the working model depicted in Fig.


These results translate into impressive numbers needed to treat of 2 for survival with mRS ≤ 4, 4 for survival with mRS ≤ 3 and 2 for survival irrespective of functional outcome.p = 0.04).Decompressive hemicraniectomy (DHC) is the most effective intervention to treat the space-occupying effect of malignant cerebral infarction. Therefore, it is recommended by international guidelines to improve survival and potentially neurological outcome in selected patients with malignant middle-cerebral-artery (MCA) infarction.Arquivos de Neuro-Psiquiatria, Rodrigues et al.In this issue ofThis highlights the importance of timely selection of malignant cerebral infarction patients requiring and potentially benefiting from decompressive hemicraniectomy.p < 0.001). Additionally, the authors calculated an infarction volume threshold for unfavorable outcome with high specificity  as > 258 ml before hemicraniectomy.Advanced cerebral imaging could be supportive for this selection process. In an analysis of the DESTINY registry including 140 malignant middle-cerebral-artery infarction patients treated with decompressive hemicraniectomy and available semiautomatic quantification of infarction, an association of infarct volume with outcome could be demonstrated. In multivariable logistic regression, beside age and stroke severity, infarct size before hemicraniectomy was an independent predictor of unfavorable outcome . The authors therefore suggested a bone deficit of at least 12 cm and additional duraplasty, as suboptimal hemicraniectomy might reduce the positive treatment effect and adversely affect functional outcome and mortality.Furthermore, beside the proper selection of malignant cerebral infarction patients with potential to benefit from decompressive hemicraniectomy, quality of the neuro-surgical procedure is crucial for the treatment effect. In an interdisciplinary neurological-neurosurgical study including 60 malignant MCA infarction patients treated with DHC, the incidence of hemicraniectomy-associated parenchymal hemorrhages and hemicraniectomy-associated infarcts was 41.6 and 28.4%, respectively.The late occurrence of the peak hemisphere volume in the study by Rodriguez et al. points at the importance of postsurgical monitoring of the decompressive effect of the hemicraniectomy. Besides neuro-radiological cerebral imaging, bedside examinations by palpation of the hemicraniectomy area, automated pupillometry and midline shift monitoring using  ultrasound could play an important role here and should be performed frequently in DHC patients. Here again, timely detection of requirement for and initiation of additional (rescue) intracranial pressure treatments as osmotic therapy or hypothermia in selected patients may be relevant for the patients' clinical course.Even though broader availability of mechanical thrombectomy for large vessel occlusion acute ischemic stroke may reduce the incidence of malignant infarctions,


Aberrant β-catenin expression and its nuclear accumulation promote the transcription of various oncogenes including c-Myc and cyclinD1, thereby contributing to tumor initiation, development, and progression. β-catenin’s expression is closely regulated at various levels including its stability, sub-cellular localization, as well as transcriptional activity. Understanding the molecular mechanisms of regulation of β-catenin and its atypical expression will provide researchers not only the novel insights into the pathogenesis and progression of cancer but also will help in deciphering new therapeutic avenues. In the present review, we have summarized the dual functions of β-catenin, its role in signaling, associated mutations as well as its role in carcinogenesis and tumor progression of various cancers. Additionally, we have discussed the challenges associated with targeting β-catenin molecule with the presently available drugs and suggested the possible way forward in designing new therapeutic alternatives against this oncogene.β-catenin is an evolutionary conserved, quintessential, multifaceted protein that plays vital roles in cellular homeostasis, embryonic development, organogenesis, stem cell maintenance, cell proliferation, migration, differentiation, apoptosis, and pathogenesis of various human diseases including cancer. β-catenin manifests both signaling and adhesive features. It acts as a pivotal player in intracellular signaling as a component of versatile WNT signaling cascade involved in embryonic development, homeostasis as well as in carcinogenesis. It is also involved in Ca It is a seminal component of the WNT signaling pathway, which is well studied, and demonstrated to be involved in the establishment of body axis and orchestration of tissue and organ development during embryonic growth [β-catenin is an 88 kDa versatile, multifunctional, and evolutionary-conserved protein known to contribute to the regulation of cell fate and development processes all through the lifespan of animals. It is identified as the c growth . Even inc growth ,3. Given2.2+dependent cell adhesion molecule uvomorulin, which was later renamed E-cadherin [Catena’ meaning chain in Latin) indicating their potential role in anchoring E-cadherin to the cytoskeleton structures.β-catenin is a well-recognized key effector of the WNT signaling pathway, which was initially discovered for its structural role in cellular adhesion. In 1989, Ozawa et al. initially investigated the structural association of β–catenin along with α-catenin and γ-catenin with the cytoplasmic domain of Cacadherin . The thrDrosophila wherein the armadillo (ARM) gene, was identified as a key segment polarity gene in early patterning and morphogenetic screens investigating mutations influencing embryonic development [ARM mutants were found to bear resemblance to Wingless (Wg) null mutant’s embryonic phenotype. Wg is a secreted extracellular protein that affects the determination of cell fate beyond its site of production along its morphogen gradient. However, the transformations seen in ARM embryos were authenticated to be cell autonomous, suggesting that ARM acted downstream to wingless. Epistatic analysis revealed that the armadillo’s segment polarity function was regulated by wingless [The potential role of β-catenin in cellular signaling was deciphered using a series of developmental studies in elopment . ARM mutwingless , which pwingless .Analysis of β-catenin conditional gain and loss of function mutant mice further revealed that canonical WNT signals control progenitor cell expansion decide lineage in the early embryo and various adult organs, thereby emphasizing its crucial role in the development as well as a disease . β-catenThe obvious question that arises is how the β-catenin molecule efficiently pivots between two distinct processes. The answer to these intricacies of multi-functionality exhibited by β-catenin lies in its structure, specific phosphorylation mechanisms, competition among interacting partners, and spatio-temporal segregation.3.ARM repeat domain, the most conserved region of the β-catenin protein. This region has 12 imperfect ARM sequence repeats (R1–12), where each ARM repeat constitutes a tandem repeat of about 42 aa forming a motif of three α-helices forming a triangular shape [ARM repeat domain. Additionally, they are also structurally flexible, and are known as intrinsically disordered protein regions (IDPRs). Though the exact mechanisms remain largely elusive, these disordered tails have unique roles in synergistically regulating the interaction patterns of β-catenin [β-catenin is composed of 781 amino acid (aa) residues having three major domains with distinctive charge distributions. The amino-terminal domain (NTD) constitutes approximately the first 130 aa residues followed by the central core region of about 550 aa forming the ar shape . The 12 ar shape . Various-catenin . It is a-catenin . Schemat4.Drosophila showed that the cell nucleus is surrounded by active DCs which predominantly direct the β-catenin degradation and thereby its nuclear access [The commitment to signal transduction is modulated by the cytoplasmic pool of free β- catenin that includes newly synthesized protein or the unbound protein released from the adherens junctions . The cytr access . HoweverThus, multiple mechanisms exist which inhibit β-catenin’s degradation, stabilizing it and promoting its nuclear translocation thereby eventually regulating its transcriptional activity. The major and most studied pathway involving β-catenin is the WNT signaling pathway.5.The WNT signaling is an evolutionarily conserved signaling which comprises canonical and non-canonical pathways that act either in a paracrine or autocrine manner. WNTs are a family of 19 secreted cysteine-rich glycoproteins of about 40 kDa that trigger intracellular signal transduction pathways upon binding to one of the 10 Frizzled (Fzd) receptors and an associated co-receptor . β-catenNotwithstanding, the mechanism of WNT stimulation results in a cytoplasmic accumulation of β-catenin which seems to be a prerequisite for its nuclear translocation. β-catenin does not contain the classical nuclear localization signal (NLS), and its import into the nucleus has been demonstrated to be independent of transport machinery-like importins or RAN-GTPase ,32. Howe2+ pathway and the Planar Cell Polarity pathway [Box 1.Once inside the nucleus, β-catenin exerts transcriptional control over a larger number of gene targets despite lacking a DNA-binding domain. It functions as a transcriptional co-activator and binds to multiple transcription factors of which the best characterized is its interaction with the members of the TCF/LEF family . The TCF pathway . Though  pathway ,42. Char6.While canonical WNT signaling is pivotal to the regulation of β-catenin and thereby significant to development, cell homeostasis, and carcinogenesis; the regulation of transcriptional activity of β-catenin is not restricted to the WNT-mediated signalosome activation alone. The linear paradigm of signaling is sometimes inadequate in explaining the change in signaling dynamics over time and more importantly crosstalk with other pathways and networks. The other mechanisms that regulate β-catenin are briefly discussed below.6.1.c-Myc, along with its dissociation from the adherens junction [Enhanced β-catenin signaling in response to EGFR-mediated tyrosine kinase phosphorylation was first reported in 1994, where EGF stimulation of epidermoid carcinoma cells showed a cytoplasmic accumulation and nuclear translocation of β-catenin followed by enhanced transcription of target genes such as junction . Since tjunction . Stimulajunction . In addijunction .Following EGF stimulation in HCT116 colon cancer cells, β-catenin was shown to interact with HDAC6 deacetylase leading to deacetylation at Lys49, inhibiting β-catenin’s phosphorylation at Ser45 and thus leading to nuclear localization . IGF-1 oApart from tyrosine kinases, the interacting proteins of β-catenin also influence the signaling capacities of β-catenin. The following section deals with the modulation in the levels of β-catenin by virtue of its competitive interaction with the tumor suppressor gene i.e. PTEN and also the structural protein i.e. γ-catenin.6.2.Caveolae are lipid rafts enriched in caveolin-1 (CAV1) exhibiting invaginations of the membrane that mediate clathrin-independent endocytosis. CAV1 regulates this form of endocytosis and has been reported to act on AXIN as well as LRP6 that promotes the cytoplasmic enrichment of β-catenin, in a cell and context-dependent fashion . Conde-PAnother competitor to β-catenin is γ-catenin/plakoglobin, a protein that shares structural homology and some functional similarity with β-catenin. In addition to the cadherins, γ-catenin has been demonstrated to bind to multiple other interactors of β-catenin including APC, GSK3β, TCF, LEF, AXIN, etc. . In thisIn essence, an elaborate understanding of β-catenin activation shows us that the transcriptional activity of the molecule is not restricted to only being a functional readout of the WNT pathway. Knowledge of the functional affiliations and crosstalk of protein is central in comprehending how the molecule is fine-tuned by cancer cells to promote proliferation, EMT, stemness, etc. In the following section, we take a glance at deregulation and its implications in malignancy.7.CTNNB1 appears to be a mutation hotspot owing to a higher frequency of missense mutations in exon 3 which encodes for the regulatory site at the amino-terminal domain of β-catenin [TP53 gene have been associated with cancer invasion by activating WNT/β-catenin signaling in mouse models of colorectal cancer [TP53 are relatively common in human cancers giving rise to mutant p53 proteins with non-operational tumor suppressive activities [TP53 mutant [CTNNB1 mutations were found to mutually exclusive to TP53 mutations indicating that either of the two mutations might be adequate to initiate the carcinogenic process [Accumulating evidence suggests an unquestionable association between aberrant β-catenin expression and cancer development, hence establishing it as a cardinal molecule in carcinogenesis. β-catenin is known to be involved in cancer progression, invasion, as well as metastasis through the regulation of its transcriptional targets, including multiple oncogenes and tumor suppressor genes. The importance of β-catenin (independent of APC) in colon carcinogenesis gained traction after the discovery of mutations in the regulatory sequences of the β-catenin gene in APC wild-type colon cancers . Mutant -catenin . Interesl cancer ,61. Misstivities ,63. Canctivities . Sandot tivities . p53 exptivities . Using ativities . In a st3 mutant . In a pa process . The eme process . The aut process . In cont process ,71. DiffCTNNB1 mutations was observed in endometrial (16%), hepatobiliary (12%), melanoma (7%), and colorectal (6%) cancers.With the advent of cancer genome databases, comprehensive and focused analysis of the mutation status of various tumor-associated genes is now possible. Large scale mutational setting of β-catenin was revealed in a Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) study employing clinical sequencing of 10,000 prospective cancer patients . A high 7.1.The role of β-catenin in carcinogenesis was initially described and intensively studied in colon cancers. APC mutations are observed in over 80% of all Colon Cancers, which invariably exhibit cytosolic and nuclear accumulation of β-catenin.CTNNB1 alterations with a high number of in-frame deletions in a targeted capture assay [In CRC, most somatic mutation hotspots are observed at Asp32, Ser33, Gly34, Ser37, Thr41, and Ser45 in exon 3 of β-catenin mRNA which stabilizes β-catenin by disturbing the phosphorylation-dependent degradation leading to carcinogenesis. Ser 45 is a priming phosphorylation site for CK1α; Ser33,Ser37, and Thr41 are phosphorylated by GSK3β, while D32 and G34 are required to bind with β-TrCP . Yaeger re assay . Activatre assay . In a sere assay . On the re assay .7.2.CTNNB1 exon 3 mutations are associated with an aggressive subtype of low-grade and low-stage endometrial carcinoma in younger women [CTNNB1 gene resulted in dysregulation of WNT/β-catenin signaling, and endometrial epithelial differentiation leading to hyperplasia in the murine model [CTNNB1 or APC genes were observed to be 30% or 12% [MYC and CCND1 were associated with worse overall survival in low-grade tumors [CTNNB1 mutations, rather than APC mutations act as direct drivers rather than passengers. A study using 245 endometrial cancer patient samples by Kurnit et al. demonstrated that mutational analysis of CTNNB1 gene could be helpful in the identification of low-grade, early-stage endometrial cancer patients with a higher risk of recurrence [CTNNB1 mutation, 42 (84%) of the patients demonstrated β-catenin nuclear staining [CTNNB1 mutant from wild type, though the sensitivity was on the lower side (84.9%). The authors recommended that immunohistochemistry could be the initial screen, and samples that are negative for β-catenin nuclear staining can be forwarded for CTNNB1 sequencing [CTNNB1 mutation in endometrial cancers [β-catenin mutations could be potential prognostic markers in endometrial carcinoma. Integrated analysis revealed that er women . It was ne model . In the % or 12% . The spe% or 12% . A separe tumors . Thus, icurrence . Among tstaining . Furtherquencing . On the  cancers .7.3.CTNNB1 gene, are relatively infrequent in breast cancer [β-catenin accumulation in the nuclei and/or cytosol of tumor cells has been observed in approximately 50–60% of human breast cancers and is associated with poor prognosis ,84. Mutat cancer . Activatt cancer ,87. Botht cancer . The gent cancer . Both get cancer ,93.The subtype-specific deregulation of WNT/β-catenin signaling may explain why some studies have failed to observe pathway activation or an association with any clinical parameters . In any WNT/β-catenin signaling is involved in the pathogenesis of various cancers ,99. This8.Various components of the β-catenin signaling pathway are under intense investigation as potential therapeutic targets owing to their significant roles in multiple malignant programs including cancer stemness and metastasis. Various strategies have been developed to date for targeting β catenin including small molecules, antibodies, RNAi, and peptides ,102. FurStarting at the extracellular level, there is an opportunity to dampen the downstream signaling by targeting the WNT ligands themselves or by antibodies that selectively bind to the extracellular domains of receptor tyrosine kinases . Restora8.1.armadillo domain where the major-binding partners include TCF3, TCF4, LEF1, BCL9, AXIN, APC, E Cadherin, etc. Structurally, two key challenges complicate the possibility of therapeutically targeting β-catenin:Overlapping PPI interfaces: A successful targeting of the β-catenin protein would entail the disruption of its interaction with its transcriptional partners while leaving the E cadherin/APC interaction unperturbed. This is because the PPI with cadherin is necessary for proper maintenance of cell–cell adhesion and epithelial phenotype while the APC interaction is crucial in moderating β-catenin degradation [radation . DisruptThe protein–protein interaction between β-catenin and the TCF/LEF members: TCF3 and TCF4 primarily bind along the positive charge groove of the armadillo superhelix [2 which is atypically large and hence a challenging hurdle. The KD of this interaction is estimated to be around 10 nM, which is significantly lower compared to catenin-cadherin and catenin–APC interactions that fall in the range of ≤82 nM and ≤ 3.1uM respectively [D makes it a nonviable target in terms of achieving inhibitor specificity and as a consensus considered a dead end in terms of inhibitor development, since any inhibitor should be able to overcome the high affinity of this bond.perhelix . The areectively . Adding To comprehend the prospects of directly targeting β-catenin, we must first re-acquaint ourselves with the dynamics of its structure. As discussed earlier, most of the protein–protein interactions (PPIs) of β-catenin happen via the 8.2.in vivo [The landscape of this so-called ‘un-targetability’ of β-catenin was changed by Shivadasani’s lab in 2004, wherein a seminal screening with about 7000 compounds was focused on achieving reproducible and dose-dependent inhibition of the β-catenin-TCF4 PPI. The screening revealed eight different compounds – PKF115–584, CGP04090, PKF222–815, PKF118–744, PKF118–310, ZTM000990, NPDDG39.024, and NPDDG1.024. Of these two compounds, CGP04090 and PKF115–584 showed limited toxicity, while inhibiting β-catenin in Xenopus embryo in vivo . A majorin vivo .A noteworthy observation concerning the β-catenin -TCF PPI is that despite the large area of interface of the two proteins, there exists a few key residues at the interface or ‘hotspots’ that contribute to the major share of the binding free energy and a mutation or disruption of the interaction of these particular residues will interfere significantly with the binding affinity of the proteins . Over thin silico docking paired with biophysical screening to look for molecules that targeted the K435 and R469 hotspot region of β-catenin -TCF PPI. About 17,700 compounds were screened of which PNU-74654 was identified as a prospective candidate [The first study that targeted the hotspot regions of β-catenin was done by Trosset et al., as they employed andidate . Furtherandidate . In a standidate . The strandidate .B was developed which displayed competitive inhibition by mimicking the alpha helical domain of BCL9 protein [TCF-dependent luciferase reporter system was used to scan a library of 22,000 compounds by Hwang et al., which resulted in the identification of MSAB . Biotin- protein . A compr9.As discussed, WNT/β-catenin plays a crucial role in the cancer progression and metastasis. Previous studies have established the positive role of WNT/β-catenin signaling in giving rise to EMT process, which is characterized by the nuclear translocation of β-catenin protein in transformed cells thereby suppressing the E-cadherin protein. This loss of E-cadherin results in the dissociation of cell–cell adhesion through which cancer cells gain motility. Various EMT-inducing transcription factors, which are downstream to β-catenin have been shown to suppress E-cadherin directly are Twist, Snail, Slug, and ZEB and are also believed to be controlled by MicroRNAs (miRNAs) in various cancers . miRNAs 10.Curcuma longa plant has been shown to possess anti-inflammatory, neuroprotective, and antioxidant activity. In cancers, curcumin has been shown to affect various oncogenic signaling, in particular WNT/β-catenin signaling. In breast cancer cells, we have previously shown that curcumin effectively impairs the expression of several WNT/β-catenin signaling components viz. E-cadherin, Dishevelled, GSK3β, β-catenin, Cyclin D1, and Slug [in vitro and in vivo by suppressing the β-catenin nuclear translocation, and interfering with β-catenin/TCF complexes to their respective DNA-binding sites. Resveratrol, a flavonoid also inhibits the β-catenin/TCF4 interactions and facilitates the TCF4 proteasomal degradation [lgs) and Pygopus I and II  [Lonchocarpus Sericeus inhibited activation of β-catenin induced through either the wild-type β-catenin, β-catenin(S33A) of dn TCF4 VP16 by impairing the TCF4 mediated transcription [Silybum marianum) extract demonstrated the downregulation in β-catenin transcriptional activity in human colorectal SW480 cells [Fraxinux rhynchophylla that has been shown to disrupt the β-catenin-TCF complex  of the β-catenin protein in colon cancer cells. Esculetin effectively antagonized the β-catenin-dependent activity and suppressed the growth in a colon cancer xenograft tumor in mouse model [et al, found that carsonic acid (from rosemary) inhibits the transcriptional β-catenin in colon cancer cells. Using NMR and analytical ultracentrifugation, the author demonstrated that carnosic acid riposte depends on an intrinsically labile α-helix (H1) amino-terminally juxtaposed the BCL9-binding site in β-catenin [β-catenin protein is now well-established as a cancer promoter, and it has been demonstrated as a potential therapeutic target for anti-cancer strategy ,135. In and Slug . Curcumiand Slug ,139. Autradation ,141. Fur pygoII) . Anothercription . The aut80 cells . Silibin80 cells . Esculetse model . In a sc-catenin . The oth-catenin ; Toxofla-catenin ; and 2-H-catenin . A compr11.The development of cancer is characterized by an abnormal and uncontrolled proliferation of cells. EMT, stemness, angiogenesis, modification in the tumor microenvironment, and immunomodulation are essential tactics employed by the cells to fuel their aggressiveness, which eventually leads to metastasis and therapy resistance. WNT/β-catenin pathway has been implicated in all these phenomena and hence put forward its significance in the context of cancer therapeutics. However, the idea that activation of the WNT pathway is restricted to a linear or sequential series of events that happens upon receptor activation by WNT ligands is an oversimplification. The pathway features a network of interconnected interactions and the present understanding of the mechano-transduction is yet to reach the temporal and molecular resolution to comprehend the exact dynamics of its complex pathophysiology. Also, though β-catenin is often (wrongly) used synonymously with the WNT pathway, as it is regulated by an array of other molecules, pathways, and network crosstalk’s that cancer cells oftentimes take advantage of this malleability to propel its growth and metastasis. From a therapeutic point of view, this interplay provides multiple potential targets for intervention.in silico screening approaches. Rational drug design has also been employed, which has led to the development of molecules like UU-T01, UU-T02, HI-B1, 4FNPC, etc., and more interestingly, β-catenin targeting peptides like NLS-Stax-h and SAH-BCL9 [Many compelling strategies have been proposed and executed starting at the level of WNT ligands, with the goal of inhibiting nuclear β-catenin and consequently its transcriptional activity. The landscape of WNT therapeutics, however, has witnessed a gradual shift in focus from upstream targeting to the targeting of downstream components or more specifically, nuclear β-catenin itself and its interaction with transcriptional partners. Traditionally, the protein has been considered undruggable due to the peculiarity of its structure but this myth of ‘undruggability’ has been challenged successfully by the design of small molecules, peptide sequences, and even natural compounds that have been demonstrated to target β-catenin PPI with its partners as discussed in the present review. Many of these molecules are identified through virtual docking experiments and other SAH-BCL9 ,151–154.12.The terminal domains of β-catenin: Most of the therapy design so far for β-catenin has been concentrated on a few hotspot regions and residues present in the central armadillo domain, while leaving the amino and carboxy domains largely unexplored due to their intrinsically disordered nature. While the terminal domains have a major functional contribution; very little is known in the way of their crystal structure because of their inherent conformational heterogeneity and highly dynamic secondary structures. Emerging strategies of in silico simulation studies and atomic characterization employing spectroscopy have the scope to provide information on their dynamic conformational ensembles. Also, the potential of stabilizing the secondary structures of these domains via small molecules/peptides cannot be disregarded either.‘Cloud’ inhibition and combination therapy: Of note, WNT pathway deregulation is accompanied by simultaneous mutations/deregulations in other pathways. Hence, targeting isolated molecules might not be significant from a therapeutic point of view as the ‘extent’ of pathway impediment or suppression of tumor progression achievable by downregulating a single molecule is debatable. A more pragmatic approach would be to target the imbalanced pathway at different points concomitantly [mitantly . This ‘CPhytochemicals and repurposed drugs: As discussed extensively in the review, manyβ-catenin targeting molecules have been identified . Natural products viz. flavonoids, sterols, lignins, and other polyphenolic compounds are shown to regulate β-catenin function by modulating the intracellular β-catenin levels through inhibition of its expression, expediting its degradation, regulating its phosphorylation status, and controlling its nuclear import. These phytochemicals offer an attractive advantage of serving as templates that can be used to streamline drug design that offers enhanced specificity and reduced toxicity. Apart from phytochemicals, drug repurposing has also gained attention considering drug discovery being time-consuming and monetarily taxing. A selected few NSAIDs , antimicrobials, antiparasitics, etc., have shown potential to modulate the WNT signaling components and they hold much prospect as well.Fundamental mechanistic studies: The solution to safely targeting β-catenin lies in unraveling the pleiotropy of the molecule. For successful translation to the clinic, fundamental studies are warranted in the following areas i.e. spatio-temporal resolution of the nuclear import of stabilized pleiotropy; tissue-specific interactors of the β-catenin protein and its influence on the transcriptional output; characterizing the interacting partners and target proteins of β-catenin that influences its role in enhancing stemness competence and plasticity of cancer cells.Our evolving understanding regarding the intricacies associated withβ-catenin structure and functions has opened several new windows of opportunity. A few plausible but relatively untraversed strategies are discussed briefly below:in vitro systems. The lifetime and efficacy along with the thermodynamics and kinetics of drug–target interactions in open/or in vivo systems vary drastically where the drug concentration and permeability fluctuate over time, affecting its in vivo potency. Acknowledging β-catenin’s role in normal cells, a safe targeting also entails minimal toxicity. To this end, future studies should also prioritize optimizing pharmacodynamic factors of the inhibitors like half-life, retention time, and renal threshold, which can have dramatic effects on the safety and efficacy of drugs.It is also worth mentioning that most of the drug target interactions of β-catenin inhibitors have so far been studied via closed/or 13.Tissue specificity of WNT pathway alterations and the possibility of a tissue-dependent predisposition in the transcriptional output of β-catenin are questions that still lack definitive answers. Thus, the versatility of β catenin has proven to be a double-edged sword due to which β-catenin targeted therapeutics have to undergo a robust clinical translation and validations. It would be interesting to explore the context-dependent differences in pharmacological and signaling dynamics brought upon by β-catenin inhibition. With enough deliberation, therapeutic targeting of β-catenin might address concerns of stemness, plasticity, and resistance of malignant cells, which remain elusive to conventional chemotherapy.


Muscular fatty infiltration is a common and troublesome pathology after rotator cuff tears (RCT), which mainly derives from fibro-adipogenic progenitors (FAPs). Compared to the RCT, fatty infiltration is not so severe in Achilles tendon tears (ATT). The knowledge of why fatty infiltration is more likely to occur after RCT is limited. In this study, more severe fatty infiltration was verified in supraspinatus than gastrocnemius muscles after tendon injury. Additionally, we revealed higher adipogenic differentiation ability of RCT-FAPs in vitro. Activation of Akt significantly stimulated GSK-3β/β-catenin signaling and thus decreased PPARγ expression and adipogenesis of RCT-FAPs, while the inhibition effect was attenuated by β-catenin inhibitor. Furthermore, Wnt signaling activator BML-284 limited adipogenesis of RCT-FAPs, alleviated muscular fatty infiltration, and improved parameters in gait analysis and treadmill test for RCT model. In conclusion, our study demonstrated that suppressed Akt/GSK-3β/β-catenin signaling increased PPARγ expression and thus contributed to excessive adipogenesis in RCT-FAPs. Modulation of Akt/GSK-3β/β-catenin signaling ameliorated excessive fatty infiltration of rotator cuff muscles and improved shoulder function after RCT. Rotator cuff tears (RCT) are common musculotendinous injuries which lead to functional disability and persistent pain for a large number of patients , 2. SecoAlthough the detailed mechanism of muscular fatty infiltration after RCT is still unknown, the role of fibro-adipogenic progenitors (FAPs) has always been emphasized. It has been reported that approximately 96% of cells which expressed adipogenic marker adiponectin in supraspinatus muscles were derived from fibro-adipogenic progenitors (FAPs) after tendon injury . Both thHowever, the in-depth and detailed mechanism underlying excessive fatty filtration caused by FAPs after RCT has not been fully clarified. Although AKT signaling and WNT5a/GSK3/β-catenin pathway have been solidly demonstrated as crucial pathways for FAPs adipogenesis –13, the Here, we found a greater propensity for adipogenesis of FAPs isolated from RCT compared to ATT. Mechanically, suppressed Akt/GSK-3β/β-catenin pathways increased PPARγ expression, leading to excessive adipogenesis of RCT-FAPs. Activation of Akt/GSK-3β/β-catenin signaling could be a therapeutic strategy to abate excessive fatty infiltration of rotator cuff muscle and improve function of shoulder joint after RCT.The Achilles tendon and rotator cuff injury mouse models were first established by transverse incision of tendon. Six weeks after tendon injury, muscle degeneration was confirmed in supraspinatus and gastrocnemius muscles Fig. . There wWe then investigated why more fatty infiltration occurred in supraspinatus muscle after tendon injury. Since FAPs were the primary source of muscular fatty infiltration after tendon injury , we focuWe then investigated whether there was a higher adipogenic differentiation potential of RCT-FAPs after tendon injury. After adipogenic induction, more lipid marked by oil red was observed in RCT-FAPs than in ATT-FAPs Fig. . ImmunofWe next performed the RNA-sequence to compare the expression profile between freshly isolated RCT-FAPs and ATT-FAPs. The differentially expressed gene analysis results were shown by heatmap and volcano chart Fig. . RNA-seqIn addition, it was worth noting that the Wnt signaling pathway, PPAR signaling pathway, and PI3K-Akt signaling pathway were highly enriched by GO and KEGG analysis Fig. . PreviouNext, we explored whether adipogenic differentiation of RCT-FAPs was regulated by Akt/GSK-3β/β-catenin pathway. SC-79 is a known Akt activator. Freshly isolated RCT-FAPs were cultured in AIM with or without SC-79 for 7 days. Oil red staining and RT-qPCR showed that activation of Akt significantly decreased the adipogenic differentiation ability of RCT-FAPs Fig. . FurtherSince our data showed reduced β-catenin activity was a key effector for increased adipogenesis of RCT-FAPs, we next investigated whether activation of β-catenin could alleviate fatty infiltration and improve shoulder function after rotator cuff injury. β-catenin activator BML-284 was used to treat RCT-FAPs in vitro. Western blotting results confirmed increased β-catenin activity and decreased PPARγ protein expression after BML-284 treatment Fig. . Oil redTo test this potential application, we conducted massive rotator cuff tears by complete transverse incision of supraspinatus and infraspinatus tendon in mice. The mice were randomly divided into two groups by SPSS software: BML-284 group received 50 ul 1 mM BML-284 injection in rotator cuff every 3.5 days for 6 weeks, while in the control group, muscular DMSO injection in rotator cuff muscle was performed with the same frequency Fig. . At the Furthermore, to evaluate the shoulder function of RCT mice after BML-284 treatment, gait analysis and treadmill test were performed. Three parameters in gait analysis were recorded. The stride length represented shoulder abduction, the stance width indicated the ability to load the limb, and the paw area reflected the degree of chronic pain , 21. SigIn the current study, there was higher adipogenic differentiation potential in RCT-FAPs than ATT-FAPs. Suppressed Akt/GSK-3β/β-catenin signaling increased PPARγ expression and thus contributed to excessive adipogenesis in RCT-FAPs. Activation of Akt/GSK-3β/β-catenin signaling could be a therapeutic strategy to abate excessive fatty infiltration of rotator cuff muscle and improve function of shoulder joint after RCT.As a common illness in orthopedic patients, rotator cuff tear always contributes to pain and functional limitations. Although surgical repair is routinely performed to treat patients with ruptured rotator cuff tendons, there is a high rate of tendon re-tearing after repair . The fatOne of the major findings of current study was that there was higher adipogenic differentiation potential in RCT-FAPs than ATT-FAPs. The distinct intrinsic differences of muscle tissues from different anatomical locations have been revealed , 17, 25.The current study also indicated that suppressed Akt/GSK-3β/β-catenin signaling increased PPARγ expression and thus contributed to excessive adipogenesis in RCT-FAPs. Previous study has demonstrated the involvement of AKT and GSK-3β/β-catenin pathways in regulating the FAPs adipogenesis. Reggio et al. revealed that inactivation of AKT/mTOR pathways suppressed the adipogenic differentiation potential of FAPs in Duchenne muscular dystrophy mice . In addiSince FAPs have emerged as a potential consequential cell source for muscular fatty infiltration , increasOne of the primary limitations of present study was that the human RCT in clinical settings was a little bit different from mice RCT model in pathogenesis, age, and anatomy. Although mice RCT model could also yield consistent outcomes in muscular fatty infiltration, the results in current study should be transferred to humans with caution. In addition, although FAPs contributed a lot to fatty infiltration after RCT, the role of adipogenesis of other types of cells should not be ignored. Thus, β-catenin agonist BML-284 might alleviate fatty infiltration by targeting other involved cell populations. Furthermore, the RCT-FAPs and ATT-FAPs investigated in current study were obtained only from one time point (one week after tendon injury). The time-dependent changes of FAPs after tendon injury should be further investigated. All issues above need to be fully addressed to thoroughly clarify the mechanism and treatment of excessive muscular fatty infiltration after RCT.In conclusion, there was higher adipogenic differentiation potential in RCT-FAPs than ATT-FAPs. Suppressed Akt/GSK-3β/β-catenin signaling increased PPARγ expression and thus contributed to excessive adipogenesis in RCT-FAPs. Modulation of Akt/GSK-3β/β-catenin signaling ameliorated excessive fatty infiltration of rotator cuff muscles and improved shoulder function after RCT.Male C57BL/6 mice (twelve-week-old) were purchased from Animal Model Research Center of Nanjing University. The RCT and ATT animal models were established as previously described –30. BrieSupraspinatus, infraspinatus, and gastrocnemius muscles were dissected one week after tendon injury. The muscles were cut into pasty and digested by collagenase II  for 45 min. Then they were further digested with the mixtures of dispase  and collagenase II for 30 min. The single-cell suspension was stained with APC-CD31 , APC-CD45 , PE-PDGFRα  for 40 min at 4 °C. Finally, CD45-CD31-PDGFRα+ murine FAPs were obtained by BD Influx sorter (BD Biosciences) [The FAPs were cultured in growth medium on Matrigel-coated  plates. The growth medium contains high-glucose DMEM , 20% FBS , 2.5 ng/ml bFGF , and 1% Penicillin-Streptomycin . 10 μM SC-79 , 1 μM BML-284  and 1 μM KYA1797K  were applied during the specific experiment. Adipogenic induction medium (AIM) was used to induce the adipogenic differentiation for 7 days . The AIMCultured cells or frozen tissue slices were fixed in 4% paraformaldehyde for 20 min, blocked with 1% BSA for 1 h and permeabilized by 0.5% Triton X-100 for 30 min. Then they were incubated with anti-PDGFRα , anti-Laminin , and anti-perilipin A/B  at 4 °C overnight. Subsequently, Alexa 488- or Alexa 594-labeled anti-mouse or rabbit secondary antibodies (Invitrogen) were used at room temperature for 1 h. The nuclei were stained by 4, 6-diamidino-2-phenylindole  for 5 min. The images were acquired by BX53 or IX73 fluorescence microscope (Olympus). All the images were analyzed by image J or image-pro plus 6.0 software.Tissue slices or cultured cells were first fixed in 4% paraformaldehyde for 10 min, followed by oil red  treatment for 10 min. Then samples were stained with hematoxylin for 5 min and mounted with 10% glycerol. For quantification analysis of lipid after adipogenic differentiation, the oil red was extracted by isopropanol for 5 min, and the value at 496 nm absorbance was detected by a microplate reader (Thermo Fisher Scientific).g for 3 min. The supernatant was used to detect triglycerides by microplate reader at 510 nm absorbance.Triglycerides colorimetric assay kit  was employed to quantify the muscular fatty infiltration after tendon injury. Briefly, the muscle was first grounded with isopropanol . Then tissue fragments were discarded by centrifugal machine at 10000 × Noldus Catwalk system was administrated to perform gait analysis after RCT. Weights were measured to avoid interference of body size. All mice walked through the tunnel of catwalk system and then the stride length, stance width, stance time, and paw area at the peak stance were recorded , 34.The treadmill test was performed by treadmill apparatus (ZII-PT/5 S). In endurance test, mice run on the treadmill at a constant speed of 20 m/min. In exhaustion test, the speed began at 16 m/min and was increased 2 m/min per 3 min. The situation that mice were incapable of running for 10 s would be identified as exhaustion.Lysed proteins with loading buffer were separated and transferred to nitrocellulose membranes according to the standard methods. Full membranes were blocked by 5% nonfat milk in TBST for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C: anti-GAPDH , anti-Akt , anti-phospho-Akt , anti-GSK-3β , anti-phospho-GSK-3β , anti-β-catenin , anti-PPARγ . Then the sample was incubated by HRP-conjugated secondary anti-rabbit or anti-mouse IgG antibodies  for one hour at room temperature. Lumi Q ECL luminescent liquid was used to capture the target strips.In total, 2 μl lipofectamine 2000  was mixed with 200 μl Opti-MEM™ I reduced serum medium  and 1 μl 100 μM β-catenin siRNA at room temperature for 20 min. The mixture was then equably added into the 12-well plate cultured with FAPs. The sequence of siRNA was listed as follow: β-catenin siRNA 5ʹ-GCAGAATACAAATGATGTA-3ʹ.Total RNA was extracted by EZ-press RNA Purification Kit  according to manufacturer’s instruction. RNA quantification was measured by NanoDrop 2000 (Thermo Fisher Scientific). One microgram total RNA was reverse transcribed into cDNA with M-MuLV Reverse Transcriptase . The products of reverse transcription were used for Real-Time qPCR. Quantitative reverse transcription PCR was performed by FastStart Universal SYBR Green Master  in the ABI Quant Studio 6 real-time qPCR system. The primers of RT-qPCR were shown in Table https://github.com/OpenGene/fastp) with default parameters [https://github.com/tanghaibao/Goatools) and KOBAS (http://kobas.cbi.pku.edu.cn/home.do) [RNA sequencing libraries were obtained by NEBNext Ultra RNA Library Prep Kit for Illumina . The paired-end RNA-seq sequencing library was sequenced by the Illumina NovaSeq 6000 sequencer (2×150 bp read length). The raw paired-end reads were embellished and quality controlled by fastp (rameters . Differehome.do) .n = 2(Zα/2 + Zβ)2σ2/(μc-μt)2, was used to calculate sample size [P < 0.05 was considered statistically significant. All the experiment results were presented by mean ± standard deviation. Technical replicates or biological repeats were introduced in the figure legend. All the experiments were assessed and analyzed by two independent investigators (H.Z. and X.S.) in a blinded manner.Statical analysis was conducted by GraphPad Prism 8  software. Student t test was used to compare quantitative parameters between two groups. The histological evaluation was used as primary outcome. The equation, ple size , and theSupplemental Figure Legend of sFigure1Figure S1


Sulfur mustard (SM) is a blister‐producing chemical warfare agent which could lead to a cascade of systemic damage, especially severe acute lung injury. Oxidative stress is considered to be vital processes for the SM toxicity mechanism. We previously proved the therapeutic effect of exosomes derived from bone marrow mesenchymal stromal cells in promoting the repair of alveolar epithelial barrier and inhibiting apoptosis. However, the key functional components in exosomes and the underlying mechanisms have not been fully elaborated. This research shed light on the function of the key components of human umbilical cord mesenchymal stem cell‐derived exosomes (HMSCs‐Ex). We noted that HMSCs‐Ex‐derived miR‐199a‐5p played a vital role in reducing pneumonocyte oxidative stress and apoptosis by reducing reactive oxygen species, lipid peroxidation products and increasing the activities of antioxidant enzymes in BEAS‐2B cells and mouse models after exposure to SM for 24 h. Furthermore, we demonstrated that the overexpression of miR‐199a‐5p in HMSCs‐Ex treatment induced a further decrease of Caveolin1 and the activation of the mRNA and protein level of NRF2, HO1 and NQO1, compared with HMSCs‐Ex administration. In summary, miR‐199a‐5p was one of the key molecules in HMSCs‐Ex that attenuated SM‐associated oxidative stress via regulating CAV1/NRF2 signalling pathway. First, the influence of HMSCs‐Ex on the survival rate and pulmonary function were assessed. Then, we evaluated whether HMSCs‐Ex alleviated SM‐induced lung injuries by suppressing oxidative stress and apoptosis. Further, we examined whether HMSCs‐Ex exerted anti‐oxidative stress and anti‐apoptotic effects through the delivery of miR‐199a‐5p in vitro and in vivo, and finally, we verified these effects via inhibiting the nuclear factor erythroid 2‐related factor 2 (NRF2) signalling pathway. This study provides solid evidence and a deeper understanding of the functional significance of HMSCs‐Ex in treating ALI.22.12. Human lung fibroblasts  were cultured in minimal essential medium alpha  supplemented with 10% exosomes‐deprived FBS (Invitrogen) obtained by centrifugation at 100,000×g at 37°C in 5% CO2. BEAS‐2B cells and HFLs cells were purchased from the Chinese Academy of Sciences and tested for mycoplasma contamination. HMSCs were donated by Origincell Technology Group Company and the HMSCs‐conditioned medium consisted of mesenchymal stem cell basal medium (Dakewe) supplemented with 5% animal serum‐free, xenogeneic‐free cell culture supplements .BEAS‐2B cells  were cultured with DMEM (Wisent) containing 10% foetal bovine serum (FBS) (Hyclone) and 1% penicillin–streptomycin (Gibco) at 37°C in 5% CO2.22 at 37°C for 48 h. Subsequently, the supernatants containing exosomes were harvested. The supernatants were centrifuged to remove cell debris at 2000g for 10 min at 4°C and subsequently passed through a 0.22 μm filter. The cleared supernatant was centrifuged at 120,000g for 70 min three times after transferred to a new glass tube. The exosomes underwent another centrifuge at 110,000g for 70 min and resuspended in PBS and then passed through 0.22 μm sterile filters. All steps were performed at 4°C. Finally, the Piercebicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific) was used to determine the concentration of exosomes. HSP70 , CD63  and TSG101  were detected by western blotting. Mimics of miR‐199a‐5p and the negative control (miR‐NC) were synthesized by Ribo Biotech and transfected into HMSCs‐Ex using the Exo‐Fect exosome reagent (System Biosciences). The exosomes were stored at minus 80°C.The exosomes were isolated and purified by differential ultracentrifugation from the supernatant of HMSCs and HFLs. Briefly, when the HMSCs and HFLs were cultured at 3–8 passages and reached 80%–90% confluence, they were cultured in the conditioned medium with 5% CO2.32O for 3–5 min. After removing the excess liquid and drying the mesh for 2 min under an incandescent lamp, the copper mesh was analysed using a Hitachi H‐7650 transmission electron microscope (Hitachi Company) at 80 kV.Briefly, 10 μL of the freshly isolated exosomes was dropped onto a copper grid covered with Formvar in chloroform at room temperature for 5 min. The excess liquid was absorbed with a clean absorbent paper. The grids were washed with sterile distilled water and subsequently stained with 1% uranyl acetate in ddH2.4To construct overexpressing CAV1 cell lines, full‐length CAV1 (NM_001172895) was cloned into a lentiviral packaging system (Obio Technology) and transfected into BEAS‐2B cell lines according to the manufacturer's instructions. To select the stable transduced cells, puromycin at the concentration of 10 μg/mL was used. GFP were used for confirmation. Cells were confirmed by fluorescence microscopy and western blotting.2.5n = 8 each) to investigate the effects of HMSCs‐Ex in improving the survival rate and respiratory function in SM‐exposed models: (i) the CTRL group, subcutaneous injection of propanediol solution (same volume as SM); (ii) the SM group, subcutaneous injection of SM; tail vein injection of PBS 30 min after the injection of SM; PBS were given on the first day and the third day two times; (iii) the HMSCs‐Ex group, tail vein injection of HMSCs‐Ex  30 min after the injection of SM; HMSCs‐Ex were given two times, that is on the first day and the third day, respectively; (iv) the NAC group, intraperitoneal injection of NAC (200 mg/kg) 30 min after the injection of SM; NAC was given once a day ; (v) and the HFLs‐Ex group, tail vein injection of HFLs‐Ex  30 min after the injection of SM. HFLs‐Ex were injected once a day on the first day and the third day. The mice were randomly distributed into the following groups (n = 8 each) to determine whether HMSC‐Ex‐derived miR‐199a‐5p was involved in SM‐induced oxidative stress reaction and apoptosis by targeting CAV1/NRF2 pathway: the CTRL group, the SM group, the HMSCs‐Ex group, the miR‐199a‐HMSCs‐Ex group  and the miR‐NC‐HMSCs‐Ex group . After SM exposure, the mice were either excised for evaluation or sacrificed by injecting 4 mL/kg chloral hydrate. After opening the chest, the lung tissue samples were collected for analysis. Saline was used to rinse the lungs and then removed immediately. Part of the samples was fixed in 4% buffered formaldehyde for histopathologic evaluation and immunohistochemistry. The other part was snap‐frozen in liquid nitrogen and stored at minus 80°C until analysis.As previously described, the concentration of SM was 40 mg/kg when used in determining the survival rate and respiratory function experiments and 30 mg/kg when conducting other experiments. Before use, SM was formulated with a propanediol solution (Sigma) and the volume of the injection was 0.05 mL/10 g in all mice. The final concentration of exosomes applied for treating lung injury was 20 mg/mL for each animal. NAC was used as a positive control in our study since it is accepted as a potential antioxidant against SM‐induced toxicity.2.6Lung tissues or cells samples were collected after multiple treatments, sonicated in cold PBS and centrifuged at 10,000 rpm at 4°C for 10 min. The supernatant was used for MDA and antioxidant enzyme assays detected with commercial kits (Jiancheng) following the manufacturer's protocols. BCA kit was used to determine the protein content.2.7SM‐induced ROS in mice and BEAS‐2B cells were detected by dihydroethidium (DHE) and DCFH‐DA staining, respectively. DHE staining in vivo was performed following the manufacturer's protocols . After the indicated administration, fresh lung tissues were harvested, and 5‐μm sections were cut in each group. Diluted with dimethyl sulfoxide (DMSO), the DHE stain solution was added to each tissue section and incubated for 30 min at 37°C in a humidified chamber. Then, a fluorescence microscope (BioTek) was used to detect red fluorescence signal. DCFH‐DA staining in vitro was conducted using a DCFH‐DA probe following the manufacturer's protocols (Beyotime). After multiple treatments, the BEAS‐2B cells were washed with PBS gently and then incubated with 10 μM DCFH‐DA in the dark for 20 min at room temperature. Then, the BEAS‐2B cells were washed with PBS again, and a fluorescent microscope (BioTek) was applied to determine the DCF fluorescence images. The ROS level was presented as fluorescence intensity per unit area.2.8Healthy adult male ICR mice (20–25 g) were purchased from the Experimental Animal Centre of Naval Medical University. Before the experiment, the mice were acclimated for 7 days. The animal were used and cared in accordance with protocols approved by the Institutional Animal Care and Use Committee of Naval Medical University. The mice were divided into five different groups randomly. The general conditions including body weight, food and water intake and condition of urination and defecation were recorded and carefully compared for 14 days. The primary endpoint of the study was the survival of the mice: treated versus untreated control.2.9g for 15 min. The supernatant was collected and transferred to new sterile Eppendorf tubes at minus 80°C. The total protein in the supernatant was measured using a BCA protein assay kit. The optical density (OD) was detected at 562 nm using amicroplate reader (BioTek).The mice were inserted with a small‐calibre cannula into the trachea and fixed by a 2/0 suture after anaesthetised with peritoneal pentobarbitone. The lungs were washed with 800 μL of PBS for three times, and the PBS was collected to sterile Eppendorf tubes at 37°C for more than 1 h. Then, the tubes were processed to centrifuge at 800For the analysis of W/D ratio, the mice chests were cut following sacrifice and lung tissues were excised. After removing blood from the surface, the wet lung tissues were weighed. The dry weight of the lung tissues was obtained after being dried in an electrothermal oven at 72°C for 72 h. The W/D ratio was calculated and used as an index to detect the lung oedema.2.10After fixation in 10% paraformaldehyde for 24 h, the lung tissues were dehydrated in serial concentrations of ethanol, dealt with the xylene clearing agent, and embedded in paraffin. The tissues were cut into sections in 5 μm and then subjected to H&E staining, followed by microscopic imaging under ×400 magnifications.The histological examinations were carefully assessed by a qualified pathologist. According to a scoring criteria published previously, scores ranging from 0  to 4 (severe) were assigned to the degree/abundance of alveolar wall thickening, alveolar oedema, inflammation and haemorrhage of alveoli and interstitium, necrosis, atelectasis, and pseudo membrane formation.2.112O2 solution for 15 min. Subsequently, the sections were washed in PBS and antigen retrieved using 0.1 M sodium citrate. After incubation with 3% bovine serum albumin (BSA) (Sigma‐Aldrich) at 37°C for 30 min, the tissues were added and incubated with antibody to CAV1 , NRF2 , and 8‐OHdG  overnight at 4°C, respectively. Then, the sections were subsequently treated with secondary antibodies at room temperature for 30 min on the next day. The nuclei were stained with 4′,6‐diamidino‐2‐phenylindole  and incubated for 10 min. The sections were evaluated with a bright‐field microscope (BioTek). Ten microscopic fields were randomly chosen and counted according to the positive number and staining intensity of each specimen.The molecular changes in CAV1 and NRF2 levels and oxidative stress index (8‐OHdG) levels were measured by immunohistochemistry. After deparaffinization, the tissue sections were soaked in a 3% H2.12A TUNEL protocol kit (Roche) was applied to detect genomic DNA breakage during apoptosis in lung tissues. Following deparaffinization and antigen retrieval, permeabilization of the lung tissue was performed with Triton X‐100 , followed by a subsequent incubation with 30 μL of TUNEL reaction mixture in the dark at room temperature for 1 h. Finally, the samples were stained with DAPI (Sigma‐Aldrich). Further, 10 fields of each group were chosen randomly under a fluorescence microscope (BioTek) and ImageJ software was used to calculate the average fluorescence intensity.2.13For the identification of HMSCs, extracellular surface proteins were detected by flow cytometry (Beckman CytoFlex). The HMSCs were subjected to antibodies against FITC‐CD44 , FITC‐CD29 , FITC‐CD73 , FITC‐CD166 , FITC‐CD34 , FITC‐CD45  and FITC‐CD11b , respectively, at room temperature for 30 min. Then, the treated cells were centrifugated and washed with PBS twice and suspended in FACS buffer for flow cytometry analysis. For the apoptosis analysis of the BEAS‐2B cells, SM‐treated cells were incubated with miR‐NC‐HMSCs‐Ex or HMSCs‐Ex or miR‐199a‐HMSCs‐Ex for 24 h. Following the instructions of an AV/PI Apoptosis Detection Kit (BD Biosciences), the collected cells were resuspended in binding buffer and incubated with Annexin V–fluorescein isothiocyanate for 15 min and PI at room temperature for 15 min in the dark. FlowJo Software was applied to measure the cell apoptosis data.2.1450 value of SM on BEAS‐2B cells, the cells were exposed to different concentrations  of SM for 30 min. Then, the cells were incubated with new culture medium for 24 h. For the assessment of the effect of HMSCs‐Ex on SM‐exposed cell viability, HMSCs‐Ex with concentrations of 1 × 109 particles resuspended in 150 μL of PBS were added to BEAS‐2B cells after exposure to SM at the concentration of 12.5 μM. After incubation for 24 h, cell viability assay was applied. For the analysis of miRNA inhibitors on cell viability, 10 inhibitors of miRNAs  and the control (miR‐NC) were synthesized by Ribo Biotech. The inhibitors were transfected into BEAS‐2B cells using lipofectamine RNAiMAX (Invitrogen) for 48 h. Then, SM was exposed to the cells at the concentration of 12.5 μM and treated with HMSCs‐Ex for 24 h. The CCK‐8 assay was conducted by co‐culturing with 10 μL of CCK‐8 solution and 100 μL of culturing medium at 37°C for 1 h. A microplate reader (BioTek) was applied to quantify the absorbance of the solution by OD values at 450 nm.The CCK‐8 (Dojindo) was applied to measure the cell viability. In brief, the cells were cultured at a density of 5000 cells per well in a 96‐well plate. To evaluate the IC2.15SM‐exposed BEAS‐2B cells were treated with HMSCs‐Ex or NAC or HFLs‐Ex for 24 h in six‐well plates as described earlier. The cells were then incubated with 50 mg/mL Hoechst 33342 (Sigma‐Aldrich) or 5 mg/mL JC‐1 (Beyotime) reaction mixture at room temperature for 20 min following the manufacturer's protocols. For the Hoechst 33342 staining experiment, the typical morphology of the cells was obtained, of which the apoptotic cells possessing the characteristic of nuclear condensation and/or fragmentation, the shrinking of cytoplasm, and membrane blebbing. For the JC‐1 staining assay, the increased green relative to red fluorescent intensity ratio representing the depolarization of the mitochondrial membrane potential was measured. Fluorescence pictures were taken by a fluorescent microscope (BioTek) and analysed using open‐source software plugins for ImageJ software.2.16https://www.targetscan.org/) was applied to predict the target gene of miR‐199a‐5p and CAV1 was selected as a candidate target. The wild‐ and mutant‐type sequences (mutation at the binding site of miR‐199a‐5p) of CAV1 3′‐untranslational region (3′UTR) were produced and amplified to construct pmir‐GlO‐CAV1 3′UTR wild‐type and pmir‐GlO‐CAV1 3′UTR mutant‐type recombinant reporter vectors, named as CAV1‐WT and CAV1‐MUT, respectively. After miR‐199a‐5p or miR‐NC and the recombinant vector (CAV1‐WT or CAV1‐MUT) were transfected to BEAS‐2B cells for 48 h, the relative luciferase activity was measured applying the dual‐luciferase assay system (Promega).The bioinformatics website  and anti‐NRF2 primary antibodies  overnight at 4°C. On the next day, the cells were washed three times with PBS and incubated in the dark with fluorescence‐labelled secondary antibodies for 1 h. After 3× PBS wash, nuclei staining with DAPI (Sigma‐Aldrich) was conducted for 5 min. For confocal imaging, the fluorescence images were obtained with a confocal laser scanning microscope (Carl Zeiss) and the fluorescence intensity was processed and analysed using ZEISS ZEN Imaging Software (Carl Zeiss).2.18The treated lung tissues or BEAS‐2B cells were lysed in radio‐immunoprecipitation assay (RIPA) buffer for 20 min, and supernatants were collected. The BCA assay kit was applied to determine the concentrations of proteins. Further, 20 μg proteins were loaded into the lanes of 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) before electroblotting onto polyvinylidene fluoride (PVDF) membranes (Bio‐Rad). After blocked with 3% BSA (Sigma‐Aldrich) at room temperature for 1 h, the membranes were incubated with antibodies against CD9 , CD61 , CD63 , CAV1 , NRF2 , HO1 , NQO1 , Histone H3  and β‐Tubulin  overnight at 4°C. After rinsing, the membranes were then incubated with a HRP‐labelled secondary antibodies that binds primary antibody for 1 h. Finally, the membranes were scanned using electrochemiluminescence  and quantified using ImageJ software.2.19TRIzol (Takara) and DNase (Invitrogen) were applied to isolate total RNA and remove genomic DNA from BEAS‐2B cells and lung samples. Following the manufacturer's instructions of the reverse transcriptase kit (Takara), cDNA was synthesized in TProfessional Thermocycler (Biometra). The primers were synthesized as follows: HO1 (Human), 5′‐CTGACCCATGACACCAAGGAC‐3′ and 5′‐AAAGCCCTACAGCAACTGTCG‐3′; NQO1 (Human), 5′‐GGCAGAAGAGCACTGATCGTA‐3′ and 5′‐TGATGGGATTGAAGTTCATGGC‐3′; NRF2 (Human), 5′‐GCCTGTAAGTCCTGGTCATCGG‐3′ and 5′‐ACTGCTCTTTGGACATCATTTCGTT‐3′; CAV1 (Human) 5′‐TGTTTTGCTCCTGATCTGA‐3′ and 5′‐TCGGGGAATTCAATACTCA‐3′; GAPDH (Human), 5′‐CGGATTTGGTCGTATTGGG‐3′ and 5′‐CTGGAAGATGGTGATGGGATT‐3′; HO1 (Mouse), 5′‐TCCTTGTACCATATCTACACGG‐3′ and 5′‐GAGACGCTTTACATAGTGCTGT‐3′; NQO1 (Mouse), 5′‐AGGATGGGAGGTACTCGAATC‐3′ and 5′‐AGGCGTCCTTCCTTATATGCTA‐3′; NRF2 (Mouse), 5′‐TCTTGGAGTAAGTCGAGAAGTGT‐3′ and 5′‐GTTGAAACTGAGCGAAAAAGGC‐3′; CAV1 (Mouse) 5′‐GGCACTCATCTGGGGCATTTA‐3′ and 5′‐CTCTTGATGCACGGTACAACC‐3′; GAPDH (Mouse), 5′‐AGGCCGGTGCTGAGTATGTC‐3′ and 5′‐TGCCTGCTTCACCACCTTCT‐3′; U6 (Human), 5′‐AAAGCAAATCATCGGACGACC‐3′ and 5′‐GTACAACACATTGTTTCCTCGGA‐3′; U6 (Mouse), 5′‐ATGGCGGACGACGTAGATCA‐3′ and 5′‐AGCTCTCGGTCATTTCTCATTTT‐3′. GAPDH was used as an internal control. qRT‐PCR was used to compare the relative mRNA expression levels with SYBR Green I system using TB Green Premix Ex Taq II (Takara) in ABI 7300 (Applied Biosystems Inc.) machine. To assess miRNA expression, TaqMan miRNA assays (TransScript) were applied to quantify relative miR‐199a‐5p expression levels, using RNU6‐1 (U6) small nuclear RNA as an internal control. A mean CT value of each sample was compared using the comparative cycle threshold (ΔΔCt) method.2.20t‐test for two groups comparisons or one‐way analysis of variance (ANOVA) for multiple groups comparisons. SPSS software Version 21.0 (SPSS Inc.) was applied for statistical analysis. Values of p < 0.01 and p < 0.001, indicated by two (**) and three (***) asterisks, were considered statistically significant differences.All data were presented in the form of the mean ± standard deviation. Statistical evaluations was performed using the Student 33.1N‐acetylcysteine (NAC) and SM + HFLs‐Ex. SM treatment induced a decrease in dietary intake and physical activity after 24 h. On the third day, severe weight and appetite reduction occurred in the SM‐treated mice. The mortality rate of mice gradually increased until the fifth day. The body weight of the mice began to increase after Day 5, and no mice died since Day 8. Compared with the control (CTRL) group, the survival rate of the SM‐exposed mice decreased significantly, while HMSCs‐Ex could reverse the decrease in the survival rate  but predominantly decreased in the HMSCs‐Ex group. In addition, the protein levels in the bronchoalveolar lavage fluid (BALF) and the wet‐to‐dry weight ratio (W/D ratio) of the lung tissues increased significantly in the SM group compared to the CTRL group, indicating increased exudation of pulmonary oedema  staining was applied to investigate the pathological lesions in lung sections. After exposure to SM, the mice in the HMSCs‐Ex group had fewer tissue lesions and perivascular infiltrates compared with mice in the HFLs‐Ex and SM groups, indicating an impressive protective effect of HMSCs‐Ex. In addition, less bleeding and oedema were also observed in mice treated with HMSCs‐Ex Figure . The hisa Figure . The tot3.2The increased production of reactive oxygen species (ROS) that induce oxidative stress and cell death through lipid peroxidation, protein oxidation, and DNA damage is one of the main mechanisms involved in SM toxicity. We investigated the effect of HMSCs‐Ex on SM‐induced intracellular ROS production using DHE staining. Fluorescence microscopy revealed an increase in ROS levels in the SM and HFLs‐Ex groups and a markedly decrease in the HMSCs‐Ex group Figure , indicat3.3p < 0.01). The result revealed that the group treated with concentration of 1 × 109 particles resuspended in 150 μL of PBS produced the most optimal effect.Using cell model of human normal lung epithelium cell line exposed to SM as previously reported,The levels of MDA, SOD and GSH in BEAS‐2B cells were detected to assess the oxidative stress damage and the anti‐oxidative enzyme production. BEAS‐2B cells were exposed to SM for 30 min and then treated with HMSCs‐Ex or NAC or HFLs‐Ex for 24 h. The MDA level Figure  significNext, the anti‐apoptotic ability of HMSCs‐Ex was evaluated using Hoechst and JC‐1 staining. The results showed that fewer apoptotic cells and less chromatin condensation were detected in the HMSCs‐Ex‐treated group Figure  comparedIn the pathogenesis of SM diseases, NRF2 is regarded as a critical transcriptional regulator of oxidative stress response.3.4Exosomes are membrane vesicles that can deliver specific functional components among which miRNA is the main form of functional RNA in exosomes.We then compared the effect of miR‐199a‐HMSCs‐Ex in alleviating oxidative responsiveness with that of miR‐NC‐HMSCs‐Ex. ROS levels were measured using a 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA) probe. The fluorescent intensity showed that DCF‐positive staining (an indicator of ROS generation) was enhanced in the SM group compared with the CTRL group; the administration of miR‐199a‐HMSCs‐Ex induced an additional decrease in the percentage of DCF‐positive cells Figure . As show3.5To explore the mechanism by which HMSC‐Ex‐derived miR‐199a‐5p suppressed oxidative stress, we examined both mRNA and protein expression levels of NRF2, HO1 and NQO1 in SM‐exposed BEAS‐2B cells from different groups using qRT‐PCR and western blot. The mRNA Figure  and protMiRNAs, downregulating gene expression at the transcriptional or posttranscriptional level, play important roles in a variety of biological processes.The miR‐199a‐5p and CAV1 targeting relationship was verified by dual‐luciferase reporter assay, as shown in Figure CAV1 inhibited the expression of antioxidant enzymes by direct interaction with NRF2 and subsequent inhibition of its transcriptional activity in BEAS‐2B cells.3.6MiR‐199a‐HMSCs‐Ex or miR‐NC‐HMSCs‐Ex was intravenously injected into mice after SM exposure to evaluate whether HMSC‐Ex‐derived miR‐199a‐5p could serve as an approach to treat lung oxidative injury induced by SM in vivo. As expected, miR‐199a‐5p expression in lung decreased during SM treatment, and miR‐199a‐HMSCs‐Ex injection efficiently reversed the change in mice lungs Figure . H&E staCAV1 and NRF2 levels showed similar pattern as in vitro experiments Figure . The res4Respiratory tract injury caused by SM is the leading cause of death from SM exposure. SM is a highly reactive bifunctional alkylating agent, rapidly reacting with a wide range of cellular components and molecular target spots. Oxidative stress, inflammatory response, alkylation of DNA and activation of proteolytic enzyme are all related to SM toxicity. NAC has a strong direct antioxidant activity and maintains the intracellular GSH level. Thus, it is recognized as the primary medicine candidate for treating pulmonary toxicity caused by SM.In the last few years, it has been proved that stem cell exosomes have beneficial effects on the treatment of acute respiratory distress syndrome (ARDS) and can exert stem cell–like effects. Previous studies found that BMSCs‐Ex had a therapeutic effect of boosting the repair of the epithelial barrier in pulmonary alveoli. However, neither the key components nor the mechanism of the actions was analysed. Exosomes mainly function by transducing components, including nucleic acids, proteins and so forth, in which miRNAs carried by the stem cell exosomes are the main components and have been confirmed to play a pivotal role in various damage repair functions of the mediating exosomes. MiRNAs modulate gene expression by suppressing the translation or stability of target gene sequencesIncreasing evidence suggests that certain miRNAs in the exosomes play a key role in the regulation of lung injury induced by oxidative stress and adjustment of the generation of ROS and endogenous antioxidant defence capabilities. For example, MSC‐derived exosomes alleviate ischemia/reperfusion injury in the lungs of mice through transporting anti‐oxidative stress and anti‐apoptotic miR‐21‐5p.As an essential component of exosomes, miR‐199a‐5p participates in many cellular activities,How does miR‐199a‐5p achieve the regulation of NRF2 molecules? We further analysed the target molecules of miR‐199a‐5p acting in the cells. Bioinformatics analysis and literature proved that CAV1 might be the target molecule on which miR‐199a‐5p took effect.CAV1 is the major resident scaffolding protein component of caveolae.In summary, our results showed that HMSCs‐Ex promoted the expression and nuclear import of NRF2 molecules through transducing miR‐199a‐5p and targeted binding with CAV1, thereby promoting the expression of antioxidant enzymes in lung cells and actively regulating SM‐induced oxidative stress.5In conclusion, we demonstrated the critical role of exosome‐mediated miR‐199a‐5p in regulating SM‐induced oxidative stress and apoptosis. We also expounded the precise mechanisms of how exosome‐mediated miR‐199a‐5p activated the CAV1/NRF2/HO1 axis and induced oxidative stress.The study not only increased our understanding of exosome‐mediated treatment for SM but also contributed to the development of the strategy for the effective treatment of SM. However, the treatment often does not take effect due to exosomal miR‐199a‐5p alone. Besides miR‐199a‐5p, many other miRNA and protein components in exosomes should also contribute to the therapeutic effects of exosomes in lung injury caused by SM. With the development of exosomes transformed through an engineering approach, further efforts are required to study the biological parameters  and the treatment potential for multicomponent transduction to maximize the potential of medicine delivery.Chuchu Gong: Methodology (lead); writing – original draft (lead). Zhengyan Gu: Formal analysis (lead); investigation . Xinkang Zhang: Investigation (lead); writing – review and editing . Qingqiang Xu: Data curation (lead); funding acquisition ; writing – review and editing (lead). Guanchao Mao: Validation (lead). Zhipeng Pei: Formal analysis (supporting); funding acquisition (supporting). Wenqi Meng: Methodology (lead). Jinfeng Cen: Visualization (lead). Jihao Liu: Formal analysis . Xiaowen He: Resources . Mingxue Sun: Conceptualization ; funding acquisition . Kai Xiao: Conceptualization (lead); funding acquisition (lead); resources (lead).This work was funded by the National Natural Science Foundation of China , Natural Science Foundation of Shanghai , the Shanghai Municipal Health Commission‐Outstanding Youth Foundation of Public Health (GWV‐10.2‐YQ48) and the Shanghai Sailing Program (21YF1457400).The authors declare that they have no conflict of interests or personal relationships that could have appeared to influence the work reported in this paper.All authors agree to be published. A preprint has previously been published .Data S1.Click here for additional data file.