FFSS-treated cultured podocytes show upregulated AKT-GSK3β-β-catenin signaling. The present study was undertaken to confirm (i) the activation of β-catenin signaling in podocytes in vivo using unilaterally nephrectomized (UNX) TOPGAL mice with the β-galactosidase reporter gene for β-catenin activation, (ii) β-catenin translocation in FFSS-treated mouse podocytes, and (iii) β-catenin signaling using publicly available data from UNX mice. The UNX of TOPGAL mice resulted in glomerular hypertrophy and increased the mesangial matrix consistent with hemodynamic adaptation. Uninephrectomized TOPGAL mice showed an increased β-galactosidase expression at 4 weeks but not at 12 weeks, as assessed using immunofluorescence microscopy  and X-gal staining . Immunofluorescence microscopy showed a significant increase in phospho-β-catenin  at 4 weeks but not at 12 weeks (p = 0.935) following UNX, and the levels of phospho-β-catenin (Ser675) did not change. In vitro FFSS caused a sustained increase in the nuclear translocation of phospho-β-catenin (Ser552) but not phospho-β-catenin (Ser675) in podocytes. The bioinformatic analysis of the GEO dataset, #GSE53996, also identified β-catenin as a key upstream regulator. We conclude that transcription factor β-catenin mediates FFSS-induced podocyte (glomerular) injury in solitary kidney.Increased fluid flow shear stress (FFSS) in solitary kidney alters podocyte function  GC), single nephron glomerular filtration rate (SNGFR), filtration fraction, and decreased hydraulic conductivity associated with glomerular hypertrophy, have been used to study hyperfiltration, leading to a detailed analysis of these parameters  mouse carries a lacZ transgene on a CD1 background . The transgene contains the lacZ gene under the control of a promoter consisting of three consensus lymphoid enhancer binding factor 1/transcription factor 3 (LEF/TCF)-binding motifs upstream of a minimal Fos promoter . This aln = 47) underwent unilateral nephrectomy (UNX) to remove the right kidney (UNX) or sham operation (Sham). The animals recovered in their cages following the surgery, and the left kidney was harvested for analysis at 4 or 12 weeks following UNX. The day prior to the harvesting of the kidneys, urine was collected for urine albumin and creatinine measurements. At the time of the tissue retrieval, an intracardiac perfusion was performed with ice cold 4%PFA in PBS (pH 7.8) for 10 min at 4.4 mL/min, followed by ice cold 18% sucrose in PBS (pH 7.8) for 10 min at 4.4 mL/min. The fixative solutions were prepared and filtered to remove small particles which otherwise may block the capillaries. The resected kidney was then incubated overnight in 30% sucrose in PBS (pH 7.8) on ice ) for 10 min and washed in a LacZ wash buffer . The sections were immersed and incubated overnight with gentle rocking in a staining solution  at 30 °C in the dark for 24 h in a humidified atmosphere [The tissue sections (8–10 μm in thickness) were fixed by immersion in 2% glutaraldehyde, 0.01% sodium deoxycholate, 0.02% IGEPAL-CA630, and 1 mM MgClThe tissue sections (4 μm) were fixed at 4 °C in 1:1 acetone and alcohol for 10–15 min, washed with PBS, and blocked in 5% donkey serum for 3 h at room temperature. The blocked sections were washed with PBS and incubated with primary antibodies for Podocalyxin , Total β-catenin , phospho-β-catenin , phospho-β-catenin , or β-galactosidase  in a blocking solution overnight at 4 °C. The tissue slices were then washed and incubated with fluorescent-tagged secondary antibody  for 1 h at room temperature and washed. The tissue sections were counterstained with Evan’s Blue for 4 min, washed, and mounted in an aqueous mounting fluid. The cultured podocytes were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature, washed in PBS, permeabilized in Triton X-100 0.1% in PBS for 10 min, and washed again. The cells were blocked in 1% goat serum/2.5% BSA in PBS for 2–3 h at room temperature, washed, and then incubated with primary antibodies in a blocking solution overnight at 4 °C, as described above for the tissue sections. The cells were washed and incubated in Alexa Fluor 488-tagged goat anti-rabbit secondary antibody (1:200 dilution) for 1 h at room temperature, washed, and mounted in an aqueous mounting fluid.Confocal microscopy with Z-stacking was performed on a Zeiss LSM 510 META microscope with an LSM 510 laser module. We obtained images at 63× magnification (in immersion oil). The z-stack depth of the confocal images is approximately 4 μm (+/− 1.5 μm) with a an optical section (z-slice) depth of 0.317 μm. We converted stacked images into maximum-intensity-projection images for analysis. The cells were treated with FFSS in triplicate, and 3–5 images were obtained per experiment. The photomultiplier tube voltage was kept constant throughout image acquisition to ensure comparable relative fluorescence intensities for all experimental conditions. Quantitative analysis of confocal microscope images was carried using the open-source Image-J/FIJI software to determine the predominant subcellular location of different phospho-β-catenin . The flu2 polystyrene flasks and first propagated in RPMI 1640 containing L-glutamine, 10% fetal bovine serum, 100 units/mL penicillin, and 0.1 mg/mL streptomycin  supplemented with 10 units/mL of γ-interferon  under permissive conditions at 33 °C, with 95% humidity and 5% CO2 [Conditionally immortalized mouse podocytes containing thermosensitive tsA58 mutant T-antigen (kindly provided by Dr. Peter Mundel) were seeded on collagen-I coated 75 cmd 5% CO2 . The cel2 for 2 h and untreated podocytes (control group) were placed in the same incubator, without exposure to FFSS. The slides were returned to the dishes for recovery up to 24 h at 37 °C under a 5% CO2-humidified atmosphere. The samples obtained from untreated podocytes at the end of the FFSS treatment, and at 2 h and 24 h following the FFSS treatment, were termed Control, End-FFSS, Post-2 h, and Post-24 h, respectively.Fluid flow shear stress was applied to the differentiated podocytes using a FlexCell Streamer Gold apparatus , as described previously ,14. FFSShttp://www.ncbi.nlm.nih.gov/geo, access on 24 July 2017) public functional genomics data repository was searched for “uninephrectomy” and filtered for “Mus musculus”, which returned 58 hits (as of 24 July 2017). We excluded results for uninephrectomy associated with ischemia-perfusion injury, diabetic nephropathy, drug treatment, and pooled samples. We identified the GEO dataset, GSE 53996, titled “Effects of high-fat induced obesity on gene expression in mouse kidney”. In the animal protocol used for obtaining this GEO dataset, male C57/BJ mice aged 6 weeks were randomly assigned to left uninephrectomy (UNX) or sham procedures and fed a high-fat diet or control chow diet. The mice were divided into 4 groups: Sham-chow, UNX-chow, Sham-HFD, and UNX-HFD. All mice were sacrificed under anesthesia at 20 weeks after surgery, and their kidneys were harvested. Microarray was performed on Agilent-026655 Mouse GE 4x44K v2 [n = 4): GSM1305179 LFD-sham_1, GSM1305180 LFD-sham_2, GSM1305181 LFD-sham_3, GSM1305182 LFD-sham_4, and UNX samples (n = 4): GSM1305183 LFD-UNX_1, GSM1305184 LFD-UNX_2, GSM1305185 LFD-UNX_3, and GSM1305186 LFD-UNX_4. The Gene Expression Omnibus  between sham and unilateral nephrectomy were submitted for the Ingenuity Pathways Knowledge Base tool . Core analysis was performed by comparing the significant genes with the IPA knowledge base, comprising curated pathways within the program on 24 July 2017. IPA predicted significant biological functions and pathways  affected in the kidney following unilateral nephrectomy. In addition, IPA was used to predict the upstream biological regulators and the downstream effects on cellular and organismal biology. In the analysis of the upstream regulators, such as transcription factors, kinases, receptors, etc., the further the activation z-score is away from zero, the more likely it is that the direction of change of the target genes is consistent with the upstream regulator being in an activated or inhibited state. The settings for the core analyses were as follows: Ingenuity Knowledge Base; Endogenous Chemicals included Direct and Indirect relationships; molecules per pathway: 35; and networks per analysis: 25. To identify over-represented canonical pathways, networks, and upstream regulators, differentially expressed genes n = 23, 20.1 ± 1.5 g) and the UNX  at the time of UNX or sham treatment at 4 weeks of age. Weight gain was lower at 4 weeks following UNX  but not at 12 weeks . Kidney weight could not be obtained due to the special perfusion protocol followed in these animals for X-gal staining at the time of euthanasia. Unilateral nephrectomy did impact growth at the early time point of 4 weeks, compared to the sham controls, but the UNX mice caught up with the sham-treated group by 12 weeks.Body weight following unilateral nephrectomy. There was no significant difference in body weight at the baseline between the sham ((b)Unilateral nephrectomy resulted in glomerular hypertrophy and increased the mesangial matrix. Quantitative morphometry was performed on the first 15 glomeruli, including 10 glomeruli from the outer cortical and 5 from the inner juxtamedullary regions, as the section was observed starting from one edge of the slide. Each glomerulus was analyzed for the glomerular area, glomerular perimeter, mesangial area, and mesangial cell count, which are shown in (c)p = 0.189) at the time of surgery. At 4 weeks following unilateral nephrectomy, the urine albumin/creatinine ratios in the sham  and UNX  animals were not different. However, the urine albumin/creatinine ratio increased significantly by 12 weeks in the UNX , compared to the sham-treated group  of TOPGAL mice. The boxplot distribution of urine albumin/creatinine ratio at the baseline and following unilateral nephrectomy is shown in Urine albumin excretion was increased in TOPGAL mice following unilateral nephrectomy. There was no difference at the baseline between the sham (24.2 ± 9.3 µg/mg Cr) and UNX TOPGAL mice n = 3 in each group) and X-gal staining (n = 3 in each group). Each animal was tested for β-galactosidase expression with only one method.Unilateral nephrectomy resulted in the nuclear translocation of β-catenin in TOPGAL mice. The β-galactosidase expression in the reporter mouse (TOPGAL) was consistent with a nuclear translocation of β-catenin. The β-galactosidase expression was assessed by two different techniques, namely, immunofluorescence (p < 0.001) at 4 weeks following UNX (p = 0.008), which was measured by a semi-quantitative score of 0–4 across all the glomeruli in the section p = 0.005) but not at 12 weeks (p = 0.935) following UNX (p = 0.170) or at 12 weeks (p = 0.197) following UNX (p = 0.027) at 12 weeks but not at 4 weeks (p = 0.111) following UNX (p = 0.006) and increased the Total β-catenin at 12 weeks (p = 0.008), while there was no change in phospho-β-catenin (Ser675). Thus, there was an increase in phospho-β-catenin (Ser552) in the kidneys following unilateral nephrectomy at 4 weeks and Total- β-catenin at 12 weeks, but not for phospho-β-catenin (Ser675). Unilateral nephrectomy resulted in increased levels of β-catenin. Confocal microscopy showed a significant increase in the phospho-β-catenin (Ser552) relative fluorescence units/area of the glomerular at 4 weeks (wing UNX . No chanwing UNX . Intereswing UNX . Unilatewing UNX . The resn = 3). At the end of the FFSS treatment, there was a significant increase in phosphorylated phospho-β-catenin (Ser552) and phospho-β-catenin (Ser675) and a significant decrease in the Total (non-phosphorylated) β-catenin in the nucleus  but not phospho-β-catenin (Ser675) . In this nucleus . Phosphop < 0.05) in the solitary kidney after unilateral nephrectomy in mice. To investigate the molecular mechanisms underlying unilateral nephrectomy, the microarray data were submitted for IPA core analysis. IPA core analysis predicted the canonical pathways and upstream regulators affected by unilateral nephrectomy. The top enriched categories of canonical pathways are listed in Bioinformatic analysis of GSE53996 identified 1722 microarray probes corresponding to 898 mapped genes that were differentially expressed . We have also demonstrated that an increase in Palb precedes the onset of proteinuria in animal models of diabetes, hypertension, radiation nephropathy, puromycin-induced nephrosis, and focal segmental glomerulosclerosis [2 or FFSS increased Palb, which could be blocked by indomethacin, indicating a role of PGE2 [The unilaterally nephrectomized TOPGAL mice showed an increased urinary albumin by 12 weeks, which was not evident at 4 weeks . Recentlclerosis ,35,36,37 of PGE2 ,38,39. T of PGE2 ,41. ThusThe TOPGAL mouse is a specific reporter strain with β-galactosidase activity, as a direct in vivo readout for activated β-catenin signaling. Using the TOPGAL mice, we demonstrated an increase in the β-galactosidase activity, as direct evidence for the in vivo activation of β-catenin following UNX. We used two different methods (described under Methods) to evaluate the β-galactosidase expression in TOPGAL and the data from each method support this finding. The increased β-galactosidase activity  was detectable at 4 weeks following UNX and returned to the baseline by 12 weeks. We anticipated a transient activation of β-catenin signaling, since it is a transcription factor, and its activation would cause progressive changes. A small but significant magnitude of increase in β-galactosidase activity at 4 weeks but not at 12 weeks in β-galactosidase expression is consistent with other reports. For example, the β-galactosidase activity in TOPGAL mice is observed during fetal and neonatal periods in the skeleton and is not detectable in mature animals, unless induced by mechanical loading or fracture in vivo ,43. In v2 (1 µM) or FFSS showed an increased phosphorylation of β-catenin (Ser552) but not β-catenin (Ser675) [β-catenin phosphorylation by casein kinase (Ser45) followed by GSK3β  directs it toward degradation through the ubiquitin-proteasome degradation pathway . In cont(Ser675) . Western(Ser675) . 2 results in the EP2-activated phosphorylation of GSK3β and increased nuclear translocation of β-catenin, followed by binding with LEF/TCF and transcriptional regulation of genes, including COX2, suggesting a role of β-catenin in skeletal physiology [Presently, we evaluated the localization and movement of phospho-β-catenin (Ser552), phospho-β-catenin (Ser675), and total β-catenin using confocal microscopy. The results  show theysiology ,48,49,50Considering its potential clinical significance, we adopted a third pre-clinical approach to corroborate our finding that hyperfiltration-induced kidney injury causes the upregulation of β-catenin signaling. To this end, we performed bioinformatic analysis using publicly available dataset in the Gene Expression Omnibus. As shown in 2 receptors are known to activate diverse signaling pathways, the activity of several proteins is likely affected due to FFSS. Of the four PGE2 receptors, only EP2 activation results in the nuclear translocation of activated β-catenin through five known intermediate mechanisms  for the progression to ESRD is still unclear, and specific treatment(s) are not available: (a) in adults following kidney donation and (b) in children born with solitary functioning kidney. We recently reviewed the accumulating evidence that living kidney donation increases the risk of ESRD in donors, despite stringent screening . The ris2 ,70,71,72


We found that lung inflammation was accompanied by decreased VE-cadherin expression and increased β-catenin activity in animal models and human pulmonary microvascular endothelial cells (HPMECs), illuminating the relationship among VE-cadherin/β-catenin complex, microvascular endothelial barrier integrity, and inflammation. Furthermore, we showed that the VE-cadherin/β-catenin complex dissociated upon lung inflammation, while Sirt3 promoted the stability of such a complex. Sirt3 was decreased during lung inflammation in vivo and in vitro. Sirt3 deficiency not only led to the downregulation of VE-cadherin but also enhanced the transcriptional activity of β-catenin that further increased β-catenin target gene MMP-7 expression, thereby promoting inflammatory factor COX-2 expression. Sirt3 overexpression promoted VE-cadherin expression, inhibited β-catenin transcriptional activity, strengthened the stability of the VE-cadherin/β-catenin complex, and suppressed inflammation in HPMECs. Notably, Sirt3 deficiency significantly damaged microvascular endothelial barrier integrity and intensified lung inflammation in animal model. These results demonstrated the role of Sirt3 in modulating microvascular endothelial barrier integrity to inhibit inflammation. Therefore, strategies that aim at enhancing the stability of endothelial VE-cadherin/β-catenin complex are potentially beneficial for preventing sepsis-induced lung inflammation.Inflammatory injury is a hallmark of sepsis-induced acute respiratory distress syndrome (ARDS)/acute lung injury (ALI). However, the mechanisms underlying inflammatory injury remain obscure. Here, we developed the novel strategy to suppress lung inflammation through maintaining microvascular endothelial barrier integrity. VE-cadherin is the main adherens junction protein that interacts with  Sepsis-induced acute respiratory distress syndrome (ARDS)/acute lung injury (ALI) is characterized by severe hypoxemia and acute respiratory failure and results in pulmonary vascular injury, edema formation, and inflammation , 2. Micrβ-catenin interacts with VE-cadherin that not only regulates microvascular endothelial barrier integrity but also participates in inflammation [β-Catenin plays a significant role in ARDS/ALI. While the inhibition of β-catenin remarkably increases animal survival in the sepsis-induced ALI model [β-catenin contributes to inflammation through mediating the downstream target gene expression in ARDS/ALI [β-catenin protein is stabilized and accumulated in the cytoplasm and further translocates into the nucleus where β-catenin binds to T-cell factor/lymphoid enhancer factor (TCF/LEF) to initiate downstream target gene expression, including matrix metallopeptidase-7 (MMP-7) [β-catenin also results in the upregulation of angiotension-2 protein, which damages microvascular endothelial barrier integrity [β-catenin and VE-cadherin interaction in regulating microvascular endothelial barrier integrity remains unclear in ARDS/ALI that warrants further investigation.As an intracellular scaffold protein, ammation , 8. β-CaLI model , the actARDS/ALI , 11. Onc (MMP-7) , 13. MMP (MMP-7) . Additiontegrity , 16. How+-dependent histone deacetylase, while Sirt3 is localized in the mitochondria. Previous studies have fully explored the role of Sirt1 in sepsis-induced ARDS/ALI, and they found that Sirt1 protects lung from inflammation [via the interaction of β-catenin and VE-cadherin.The emerging roles of the Sirtuin (Sirt) family in sepsis-induced ARDS/ALI have attracted extensive attention –19. The ammation –23. Sirtammation . Sirt3, ammation –26. Howe−/− mice were purchased from Cyagen Biosciences . Mice were intraperitoneally injected with sodium pentobarbital  for anesthesia. The complete CLP model was carried out by perforating the ligation end and squeezing out feces as previously described [E. coli 0111:B4, Sigma-Aldrich, USA) in sterile saline by intraperitoneal injection to induce ALI, while control mice were administrated sterile saline by intraperitoneal injection. Before CLP surgery or LPS injection, mice were administered 3-TYP  or ICG-001  in sterile PBS by intraperitoneal injection for 2 days. Mice were sacrificed at 3 h, 6 h, and 24 h after CLP surgery or LPS injection. The lung tissues were immediately frozen and stored in liquid nitrogen for subsequent experiments.The animal study was approved by the Ethics Committee of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences , and was reported in compliance with the ARRIVE guidelines . Male C5escribed , while sα) from LPS- or CLP-induced ALI mice were measured employing a commercially available ELISA kit and used according to the manufacturer's kit protocols (R&D Systems). Protein concentrations were measured spectrophotometrically at 450 nm and determined by establishing a standard curve by employing standard proteins.Serum interleukin-6 (IL-6) and tumor necrosis factor-α . The concentrations of 3-TYP and ICG-001 were both 10 μM. After 12 h or 24 h treatment, HPMEC cells were harvested for subsequent experiments.Human pulmonary microvascular endothelial cells (HPMECs) were purchased from the China Center for Type Culture Collection. HPMECs were cultured in Endothelial Cell Medium  supplemented with 10% fetal bovine serum at 37°C with 5% carbon dioxide. HPMECs were treated with 0.5 ng/mL or 1.0 ng/mL LPS (The plasmid expressing full-length human Sirt3 cDNA (Sirt3 over), the plasmid containing empty plasmids (vector), the plasmid expressing shRNA against human Sirt3 (Sirt3 shRNA), and the plasmid containing scramble (scramble) were constructed by GeneChem . HPMECs were seeded in a 6-well plate with 70-80% confluence. The plasmid and Lipofectamine 3000  were directly added to the medium, and the medium was removed after 24 h treatment according to the manufacturer's guide.μm. Hematoxylin and eosin (HE) staining was performed by following the method routinely used in our laboratory [Briefly, lung tissues were fixed in 4% paraformaldehyde at room temperature for 24 h. After processing for paraffin embedding, lung tissues were sectioned at 5 boratory . The imaboratory , 31.2O2, incubated with 10% goat serum at room temperature for 1 h, and then incubated with primary antibodies at 4°C overnight. After incubating with secondary antibodies, the slides were developed by 3,3-N-diaminobenzidine tetrahydrochloride. The slides were then counterstained with Harris hematoxylin and mounted on neutral gum. The images were acquired with a Leica microscope  and analyzed using Image-Pro Plus 6.0 software according to the mean optical density of positively stained areas in a standard protocol [Paraffin-embedded mouse lung sections and immunohistochemical staining for lung tissues were performed as described previously . After hprotocol , 33. To Briefly, HPMECs were cultured on coverslips and fixed with 4% paraformaldehyde at 4°C. The slides were incubated with 10% goat serum for 1 h and with primary antibodies at 4°C overnight and then incubated with secondary antibodies for 2 h and incubated with propidium iodide for 10 min. The slides were mounted with 80% glycerinum in sterile PBS and measured by a laser-scanning confocal microscope . To clearly present the results, the value of protein expression in the control group was further normalized to 1 using the mean.β-catenin , dephosphorylated active β-catenin , Sirt3 , matrix metallopeptidase-7 , and cyclooxygenase-2 . α-Tubulin was purchased from the Proteintech Company . The secondary antibodies of goat antirabbit  or goat antimouse  were used. PVDF membranes were visualized using a chemiluminescence Western blotting detection reagent. The signal intensity of each immunoblot was analyzed using ImageJ software , and each band density was normalized by the α-tubulin protein expression level. To clearly present the results, the value of protein expression in the sham-operated or control group was further normalized to 1 using the mean.Protein expression was examined by Western blot as previously described . HPMECs β-actin and determined by the ΔΔCt method.The mRNA expression was analyzed by qRT-PCR as previously described . TRIzol β-catenin. The lysates of HPMECs or lung tissues were pretreated with protein A/G  at 4°C for 1 h. The supernatant was incubated with anti-β-catenin (1 : 100) antibody overnight at 4°C and then immunoprecipitated by protein A/G overnight at 4°C. After washing six times, the complexes were analyzed by Western blot and the total protein was used as the input.Co-IP was used to analyze the interaction of VE-cadherin and TCF/LEF reporter assay was carried out by the procedures described previously . The Cigμg anti-β-catenin antibody at 4°C overnight, and the immunoprecipitation incubated with normal rabbit IgG was used as a control. qRT-PCR was used to amplify MMP-7, and the primer sequence is listed in ChIP assay was performed using the Pierce Agarose ChIP Kit  according to the manufacturer's instructions. The DNA/protein complexes were crosslinked in 1% formaldehyde  for 10 min. The chromatin in the nuclei obtained from cells or tissues were digested by micrococcal nuclease at 4°C for 5 min. The lysates were incubated with 3 β-catenin antibodies at 4°C overnight and incubated with PLA probes at room temperature for 1 h and then at 37°C for 2 h. The amplifications were performed at 37°C, and HPMECs were then stained by DAPI for 5 min. The cells were examined by a laser-scanning confocal microscope . To clearly present the results, the value of protein expression in the control group was further normalized to 1 using the mean.PLA was carried out using the Duolink® In Situ PLA Kit  according to the manufacturer's protocol as previously described . Brieflyt-test; statistical analysis for three or more groups was performed using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test when F achieved p < 0.05 and there was no significant variance in homogeneity. To avoid unwanted sources of variation, results were normalized to control. p < 0.05 was considered significant. The number of replicates was 6 per group for each data set.The results were expressed as mean ± SE, and GraphPad Prism software version 6.0  was employed for statistical analysis. Statistical analysis for two groups employed by Student's α levels in the LPS group were significantly elevated in a time-dependent manner (To investigate the role of Sirt3 in maintaining lung microvascular endothelial barrier integrity, we first established LPS- and CLP-induced ALI mouse models. As shown in t manner . Similart manner , indicatβ-catenin and VE-cadherin maintains the adherens junctional integrity, while the breakdown of the adherens junction releases β-catenin from the cytomembrane [β-catenin was activated during ALI. As shown in Figures β-catenin protein significantly increased in the nucleus of lung tissues in LPS- and CLP-induced ALI models in a time-dependent manner, indicating the activation of β-catenin during ALI. As shown in Figures β-catenin activity in HPMECs in dose- and time-dependent manners, which confirmed the positive correlation between β-catenin activity and ALI. Since the activation of β-catenin contributes to the destruction of the microvascular adherens junction directly through increasing downstream gene MMP-7 expression to damage the adherens junction [β-catenin, the Wnt family. As shown in β-catenin. And these results were consistent with previous studies [β-catenin during ALI was accompanied by inflammation and damaged microvascular endothelial adherens junction.The interaction of membrane . Therefojunction , 39, 40, studies . In the β-catenin were investigated using the Co-IP method. As shown in Figures β-catenin obviously lowered after LPS treatment or CLP surgery in a time-dependent manner. The weakened interaction of VE-cadherin and β-catenin may contribute to β-catenin accumulation in the cytoplasm and translocation into the nucleus to further trigger its transcriptional activity of MMP-7. As shown in β-catenin in HPMECs during lung inflammation. LPS stimulation also resulted in the significant upregulation of the MMP-7 promoter (β-catenin activation that further increased MMP-7 expression to aggravate the breakdown of the microvascular adherens junction. Our further experiments also confirmed these hypotheses. Treatment with the β-catenin inhibitor ICG-001 significantly decreased serum IL-6 and TNF-α levels (Figures β-catenin activation was positively related to inflammation in ALI. Notably, treatment with the Sirt3 inhibitor 3-TYP significantly increased serum inflammatory factor levels in LPS- and CLP-induced ALI models, indicating that Sirt3 was negatively related to lung inflammation.Next, the interactions of VE-cadherin and promoter . These r Figures , indicatAlthough we determined the relationship between inflammatory injury and Sirt3, the expression of Sirt3 during ALI remained unclear. Hence, we investigated the expression of Sirt3 protein in ALI mouse models and HPMECs. As shown in β-catenin, plasmids expressing Sirt3 shRNA, scramble, Sirt3 over, and vector were employed. As shown in Figure β-catenin and VE-cadherin expression. As shown in β-catenin upregulation and VE-cadherin downregulation after being transfected with the Sirt3 shRNA plasmid, while Sirt3 overexpression by the Sirt3 over plasmid inhibited β-catenin protein expression and promoted VE-cadherin protein expression in HPMECs (Figures β-catenin and the VE-cadherin protein. As shown in Figures β-catenin and the VE-cadherin protein in HPMECs. Sirt3 deficiency weakened their interaction while Sirt3 overexpression strengthened their interaction (Figures To decipher the role of Sirt3 in the interaction of VE-cadherin and  Figures . Co-IP r Figures .β-catenin transcriptional activity and Ang-2 expression that contributes to the breakdown of the microvascular endothelial adherens junction. While Sirt3 deficiency elevated the luciferase activity of the TCF/LEF reporter induced by LPS, Sirt3 overexpression suppressed the luciferase activity of the TCF/LEF reporter (β-catenin transcriptional activity. Additionally, Sirt3 deficiency increased the expression of MMP-7 promoters and Ang-2 mRNA expression, while Sirt3 overexpression decreased the expression of MMP-7 promoters and Ang-2 mRNA expression in HPMECs (Figures β-catenin and VE-cadherin in the cytomembrane using in situ PLA. Compared with the control group, distinct β-catenin-VE-cadherin signals were lowered upon LPS stimulation, indicating the weakened interaction of β-catenin and the VE-cadherin protein by LPS stimulation (β-catenin and the VE-cadherin protein in vitro.We further investigated the effects of Sirt3 on reporter , indicat Figures . Further Figures . We alsomulation . Of notemulation . Taken t−/− mouse model. As shown in β-catenin and MMP-7 significantly increased in the Sirt3−/− mouse. Similar results were also found in the CLP model. Compared with the wild type mouse, the downregulation of VE-cadherin and the upregulation of β-catenin and MMP-7 were observed in the Sirt3−/− mouse after CLP surgery (α levels in the Sirt3−/− mouse were higher than those in the wild type mouse in the LPS- or CLP-induced group (Figures in vivo. HE staining and immunohistochemical staining of COX-2 also showed that lung inflammation in the Sirt3−/− mouse was worse than that in the wild type mouse (β-catenin and the VE-cadherin protein (To confirm the role of Sirt3 on the microvascular endothelial adherens junction integrity to inhibit lung inflammation, we used the Sirt3 surgery . In addi Figures . These rpe mouse . These r protein .Sepsis leads to high mortality and morbidity due to multiple organ dysfunction including ARDS/ALI that causes severe systemic inflammatory response syndrome. The microvascular endothelial barrier dysfunction, microvascular permeability imbalance, and capillary leakage induced by sepsis play important roles in the occurrence and development of organ dysfunction , 43. Theα levels, indicating the occurrence of systemic inflammation. Pharmacological inhibition of Sirt3 by 3-TYP treatment or deletion of Sirt3 by gene editing technology significantly amplified systemic inflammation in vivo. These results indicated the effects of Sirt3 against inflammation in ALI. Additional experiments elucidated that Sirt3 maintained the microvascular endothelial adherens junction in HPMECs. Sirt3 overexpression significantly protected the adherens junction of the lung microvascular endothelial barrier in HPMECs, while Sirt3 deficiency promoted the breakdown of the adherens junction of the lung microvascular endothelial barrier. These results confirmed the protective role of Sirt3 on maintaining the adherens junction of the lung microvascular endothelial barrier to attenuate inflammation, which first revealed that Sirt3 suppressed ARDS/ALI-induced inflammation through maintaining the adherens junction of the lung microvascular endothelial barrier.In the present study, we identified Sirt3 as a protective factor for the adherens junction of the lung microvascular endothelial barrier. Sirt3, the main mitochondrial molecule in the Sirt family, alleviated ARDS/ALI through regulating cellular bioenergetics, mitochondrial metabolism, and inflammatory responses , 46, 47.β-catenin. The dimerization of VE-cadherin occurs in the cytomembrane, and the dimerization of the adjacent cellular VE-cadherin dimer forms cadherin clusters. The cytoplasmic domain of VE-cadherin contains the binding sites of the VE-cadherin/β-catenin complex. In the physiological status, VE-cadherin and β-catenin existed as a complex and suppressed β-catenin transcriptional activity. However, once microvascular endothelial cells were injured, the VE-cadherin/β-catenin complex dissociated and the VE-cadherin-dominant intercellular adherens junction became damaged, which caused β-catenin accumulation in the cytoplasm and translocation into the nucleus to initiate β-catenin transcriptional activity [Furthermore, we explored the underlying mechanism of Sirt3 on maintaining the adherens junction of the lung microvascular endothelial barrier. The integrity of the adherens junction involves the interaction between VE-cadherin and activity .β-catenin activity in the ALI model. Sirt3 overexpression strengthened the interaction of VE-cadherin and β-catenin, while Sirt3 deficiency weakened their interaction. Additionally, Sirt3 also regulated β-catenin transcriptional activity. Accompanied by β-catenin released from the complex, sepsis-induced ALI resulted in the upregulation of Wnt ligands that further accumulated β-catenin in the cytoplasm and translocated into the nucleus, all of which promoted β-catenin transcriptional activity. MMP-7, the downstream target gene of β-catenin, participates in the breakdown of the intercellular adherens junction that ultimately results in microvascular leakage and inflammation aggravation [in vivo and in vitro. Sirt3 also induced Ang-2 expression to damage the lung microvascular endothelial barrier. These results indicated the underlying mechanism of Sirt3 on maintaining the adherens junction of the lung microvascular endothelial barrier by acting on the interaction of VE-cadherin and β-catenin (In the present study, we found that the protein expression of Sirt3 was positively related to VE-cadherin expression but negatively related to ravation . Here, t-catenin .via the VE-cadherin/β-catenin complex. We identified for the first time Sirt3 as the crucial mediator of sepsis-induced lung microvascular endothelial barrier integrity. In summary, the present study provides novel insights into the mechanism of Sirt3 against lung inflammatory injury. We identified Sirt3 as a modulator of the adherens junction in the lung microvascular endothelial barrier to suppress inflammation through regulating the interaction of VE-cadherin and β-catenin. These results provide the potential therapeutic target and novel strategy to prevent inflammation in ARDS/ALI.Although Sirt3 is known to modulate metabolism to suppress inflammation in ARDS/ALI, we first focus Sirt3 on the microvascular endothelial barrier


Toxicities such as myelotoxicity, cardiotoxicity, genotoxicity, and vasospasm are the major side effects for common anticancer drugs and are well documented. There is, therefore, a need to develop new, effective, safer, and more affordable anticancer drugs from alternative sources. In recent years, plant-derived Chinese medicine monomers have drawn increasing attention due to their high safety, low toxicity, minimal side effects, and antitumor effects. Plant-derived Chinese medicine monomers are effective against multiple targets and can regulate the growth, proliferation, apoptosis, invasion, and migration of OC as well as reverse drug resistance by regulating the Wnt/β-catenin signaling pathway. In this review, we summarize and provide mechanisms and prospects for the use of plant-derived Chinese medicines for the prevention and treatment of OC.Ovarian cancer (OC) is a common malignant tumor of the female reproductive system and has a high morbidity and mortality rate. The progression and metastasis of OC are complex and involve multiple signaling pathways. The Wnt/ Ovarian cancer (OC) is one of the most common gynecological malignancies and is a serious threat to women's lives and health. OC is insidious, with no typical symptoms in the early stage, and most patients present with stage III/IV disease at the time of diagnosis . There wβ-catenin, JAK/STAT, PI3K/AKT, and NF-κB signaling pathways Rheum palRabdosia rubescens, and it has various biological and pharmacological activities, including antitumor, antimicrobial, and anti-inflammatory effects [in vitro experiments have shown that it has a significant inhibitory effect on more than 20 cancer cell lines [μmol/L, 10 μmol/L, and 20 μmol/L of ORI for 24 h increased the expression of E-cadherin and decreased the expression of vimentin, β-catenin, c-Myc, and cyclin D1 in a dose-dependent manner. This suggests that ORI might inhibit the Wnt/β-catenin signaling pathway and thereby inhibit the expression of related cytokines.Oridonin (ORI) is an ent-kaurene tetracyclic diterpenoid compound isolated from  effects . In recell lines , 79. Liull lines  exploredμmol/L, 20 μmol/L, and 50 μmol/L of schisandrin B for 48 h inhibited the proliferation of SKOV3 cells in both a time- and dose-dependent manner. Schisandrin B can also block cell cycle progression and reduce the protein expression levels of β-catenin, c-Myc, and cyclin D1. These results suggest that schisandrin B may reduce the proliferation of SKOV3 cells and block cell cycle progression by inhibiting the Wnt/β-catenin signaling pathway.Schisandrin B is extracted from the Chinese medicine Schisandra, and it has pharmacological effects such as promoting tumor cell apoptosis, reducing inflammation and tissue edema, improving microcirculation and antioxidation, and expanding blood vessels , 81. Zenμmol/L of apigenin for 24 h could effectively inhibit the migration and invasiveness of HO8910 OC cells, and it can also downregulate β-catenin and E-cadherin, which are the downstream effectors of the Wnt signaling pathway. The expression level modulation of genes and proteins may therefore be achieved by inhibiting the Wnt/β-catenin signaling pathway. Indeed, cytological analysis, western blotting, and immunofluorescent staining all suggest that apigenin induces autophagy-mediated downregulation of β-catenin in treated cells, thereby inhibiting the Wnt/β-catenin signaling pathway [Apigenin is a flavonoid derived from vegetables, fruits, tea, and beans , and it  pathway .Camellia sinensis) flower saponins (TFS) has antiallergic and antitumor effects [μg/ml, 3.0 μg/ml, 3.5 μg/ml, and 4.0 μg/ml reduced the viability of OCSLCs compared with the control group. TFS inhibited clonal expansion, and tumor sphere formation reduced the cells' self-renewal capacity and was shown to downregulate the expression of p-AKT, p-GSK-3β, β-catenin, and c-Myc proteins while upregulating the phosphorylation of β-catenin thereby inhibiting the Wnt/β-catenin signaling pathway. It is therefore suggested that TFS can inhibit the growth and proliferation of OCSLCs and reduce their stem-like characteristics through inhibition of the Wnt/β-catenin signaling pathway.Tea  and Western blot confirmed that the compound could downregulate the protein expression of β-catenin, c-Myc, and cyclin D1. These results suggest that icariin can inhibit the proliferation of human CAOV3 cells and that this might be achieved by inhibiting the Wnt/β-catenin signaling pathway.Icariin is the principal active ingredient in the Chinese medicine Epimedium. As a new type of flavonoid anticancer drug, it has demonstrated significant antitumor effects . Chen etβ-catenin signaling pathway-related gene expression in the cells. They found that EGCG had a strong antiproliferative effect and that 40 μg/ml of EGCG the cell cycle of HO8910 cells was completely blocked. At the same time, EGCG could significantly reduce the level of β-catenin and cyclin D1 mRNA and protein. These results suggest that the mechanism through which EGCG inhibits the growth of HO8910 OC cells may be related to the inhibition of the Wnt/β-catenin signaling pathway.Epigallocatechin-3-gallate (EGCG) is extracted from green tea and has been shown to have multiple effects on both pathological and physiological processes in humans . In receβ-catenin and c-Myc proteins. The results of a scratch test showed that the migration ability of the A2780 cells decreased significantly proportionally to the drug concentration and exposure time. These results confirmed that PAE inhibits the Wnt/β-catenin signaling pathway by regulating the expression of related proteins, thereby inhibiting the growth of A2780 cells and inducing apoptosis [Paeonol (PAE) is one of the active components of Cortex Moutan, which has various anti-inflammatory, antioxidant, and antitumor effects . Studiespoptosis , 64.Stephania tetrandra S. Moore, [μmol/L of TET inhibited the migration and invasion ability and decreased the levels of MiR-21, p-GSK3β, β-catenin, N-cadherin, and vimentin, whereas for ES-2 ovarian clear cancer cells, 3.0 μmol/L TET could do the same. Meanwhile, the E-cadherin protein expression level was significantly increased. Similarly, Jiang and Hou [β-catenin/c-Myc/cyclin D1 signaling pathway.Tetrandrine (TET) is a Chinese medicine isolated from the root of . Moore, . Modern . Moore, . Wang et. Moore,  found th and Hou  also fouμg/ml of PC extracted from the bayberry leaf could inhibit β-catenin, cyclin D1, and c-Myc protein expression, thereby impeding the self-renewal ability of drug-resistant OVCAR3 OC cells, weakening their stem cell characteristics, blocking the cell cycle, and reversing drug resistance. The suggested mechanism for these effects is related to inhibition of the Wnt/β-catenin signaling pathway.Proanthocyanidins (PCs) are commonly found in plants such as metaplasia, grape seeds, pine bark, and sorghum bark . PCs areβ-catenin in SKOV3/CDDP cells and partially reverse cisplatin resistance. The mechanism of this activity may be related to the Wnt/β-catenin signaling pathway. In addition, the combination of naringin with cisplatin might prevent cell cycle progression, thereby inhibiting the proliferation, invasion, and migration of OC cells [Naringin is a natural flavonoid that mainly exists in the peel and pulp of grapefruit and lime and has exhibited antioxidative, anti-inflammatory, and antitumor effects , 97. NarOC cells .β-catenin pathway is an important target for treating OC, and many studies have investigated the potential therapeutic effects of antibodies and small molecules that target this pathway, with some currently being tested in clinical trials [β-catenin signaling pathway to inhibit the metastasis and proliferation of OC.The Wnt/l trials . In recein vitro cultured cells. Regarding the cytotoxicity of plant-derived Chinese monomers, their bioavailability is not very clear, and further in vitro, in vivo, and clinical studies are needed. The anticancer effect of plant-derived Chinese monomers is multitargeted. At present, the most common problems in research on the antitumor effects of plant-derived Chinese monomers are insufficient validation of findings and the scarcity of clinical trials. Therefore, the anticancer effects of these monomers should be further demonstrated at different research levels. Carrying out clinical trials for plant-derived Chinese monomers with definite curative effects that are both stable and obvious will promote the translation of experimental research results into clinical practice and lay a solid modern medical theory foundation for the clinical application of plant-derived Chinese monomers against cancer. Thus, the specific mechanisms through which plant-derived Chinese monomers influence the Wnt/β-catenin signaling pathway require deepened studies in the future.Plant-derived Chinese monomers play a role in the development of new targeted therapies for the prevention and treatment of OC, but there are still some limitations to these studies. Although some mechanisms of plant-derived Chinese monomers in antitumor therapy have been discussed, the research on plant-derived Chinese monomers' anticancer effects and their ability to reverse transformation therapy resistance are mostly based on experiments involving


There is an urgent need to identify potential targets in anticancer therapy to improve the survival and prognosis of patients with ovarian cancer (OC). Herein, we investigated the functional significance of chemokine (C-X-C motif) ligand 14 (CXCL14) in OC cell growth and epithelial–mesenchymal transition (EMT).qRT PCR and western blotting was used to detect CXCL14 mRNA level and protein expression, respectively. The functional mechanism of CXCL14 in OC was investigated by CCK-8, colony formation and transwell assays. The migration ability of OC cell was determined using wound healing. The protein expressions of CXCL14 and β-catenin in OC tissues were determined by immumohistochemical staining.We demonstrated that high levels of CXCL14 were associated with a worse prognosis in patients with OC. CXCL14 knockdown considerably restrained the growth, migration and invasion of OC cell in vitro. In contrast, ectopic CXCL14 overexpression yielded the opposite results. Investigations to determine the underlying molecular mechanisms revealed that the Wnt/β-catenin signaling pathway is involved in CXCL14-facilitated OC cell invasiveness.These data collectively demonstrate that CXCL14 contributes to OC cell growth and metastatic potential by regulating the Wnt/β-catenin signaling pathway. Ovarian cancer (OC) is the most lethal cancer among gynecologic malignancies . DespiteCXCL14 is a key homeostatic chemokine that facilitates immune surveillance of skin epithelium through recruitment of various immune cell types, including natural killer cells, dendritic cells (DCs), and T cells . A studyEarlier investigations have closely linked chemokines and their membrane receptors to the pathological EMT process . For insIn this study, we evaluated the expression of CXCL14 in patients with OC and determined its correlation with the clinical outcomes of patients. Furthermore, we explored the functional significance of CXCL14 expression in OC cell invasion and EMT.2. The inhibitor of Wnt/β-catenin signaling, XAV939, was purchased from Selleck Chemicals .The human ovarian surface epithelial cell line HOSEpiC and four ovarian carcinoma cell lines  were purchased from Nanjing KeyGen Biotech . Cells were cultured in DMEM  supplemented with 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin  at 37 °C in an incubator with 5% COn = 50) and non-tumor ovarian tissues  were acquired from OC patients or patients with benign epithelial ovarian tumor, respectively . A. A20]. AEmerging evidence has established that CXCL14 is important for cell growth, invasion, metastasis, and chemoresistance in multiple cancers . AlthougCancer metastasis is a complicated process, and cancer cells need to acquire metastatic characteristics, such as decreased adhesiveness and increased migration and invasive ability, to escape the confines of the primary tumor site and establish distant metastases . In addiImportantly, Wnt/β-catenin signaling was involved in the CXCL14-induced EMT process. First, the present findings revealed that reduced CXCL14 expression obstructed EMT, whereas upregulation of CXCL14 contributed to EMT, thus indicating that CXCL14 acts as a crucial regulatory factor of EMT in OC cells. Moreover, β-catenin is a vital element in the Wnt signaling pathway, and β-catenin facilitates the transcriptional activation of EMT-associated genes, including cyclin D1, MMP-7, and Axin2. Additionally, CXCL14 knockdown in SKOV3 cells lowered the expression of Wnt/β-catenin-associated genes, whereas CXCL14 overexpression in OVCAR3 cells augmented their expression. To verify whether CXCL14 triggers OC cell growth and EMT via the Wnt/β-catenin signaling pathway, OC cells were treated with XAV939. Intriguingly, inhibition of Wnt/β-catenin signaling attenuated CXCL14-induced OC cell EMT, as well as cell growth and invasion. Thus, our data suggest that Wnt/β-catenin signaling is necessary for CXCL14-facilitated OC cell growth and invasion.In tumor tissue cells, owing to various factors, the activation of signal transducer and activator of transcription 3 (STAT3) appears to be in a persistently and abnormally high expression state, which leads to abnormal proliferation of malignant tumor cells. Previous studies have shown that overexpression of CXCL14 increases the phosphorylation level of STAT3 in OC cells, thereby promoting the activation of the STAT3 signaling pathway , 25. AccIt is well recognized that nuclear β-catenin is associated with transcription factor 4 (TCF4) to activate target gene transcription . The keyAltogether, our study demonstrates that high CXCL14 expression is associated with increased lymph node metastasis and worse prognosis in patients with OC. In conclusion, we found that CXCL14 promotes OC cell EMT and the metastatic phenotype by influencing the Wnt/β-catenin signaling pathway Fig. .Fig. 6Sc


This work involves the synthesis of hybrid oligomers based on the epoxy methacrylate resin. The EA resin was obtained by the modification of industrial-grade bisphenol A-based epoxy resin and methacrylic acid has been synthesized in order to develop multifunctional resins comprising both epoxide group and reactive, terminal unsaturation. Owing to the presence of both epoxy and double carbon–carbon pendant groups, the reaction product exhibits photocrosslinking via two distinct mechanisms: (i) cationic ring-opening polymerization and (ii) free radical polymerization. Monitoring of EA synthesis reactions over time using PAVs, MAAC and NV parameters, and the FT-IR method reveals that esterification reactions proceed faster at the start, exhibiting over 40% of conversion within the initial 60 min, which can be associated with a relatively high concentration of reactive sites and low viscosity of the reaction mixture at the initial reaction stage. With the further increase in the reaction time, the reaction rate tends to decrease. The control of the EA synthesis process can guide how to adjust reactions to obtain EAs with desired characteristics. Based on obtained values, one can state that the optimum synthesis time of about 4–5 h should be adopted to prepare EAs having both epoxy groups and unsaturated double bonds. The structure of the obtained EA was confirmed by FT-IR and NMR methods, as well as the determination of partial acid value and epoxy equivalent. Samples at various stages of synthesis were cured with UV radiation in order to study the kinetics of the process according to cationic and radical polymerization determined via photo-differential scanning calorimetry (photo-DSC) and real-time infrared spectroscopy (RT-IR) and then the properties of the cured coatings were tested. It turned out that the cationic polymerization was slower with a lower conversion of the photoreactive groups, as compared to the radical polymerization. All the obtained EA coatings were characterized by good properties of cured coatings and can be successfully used in the coating-forming sector. Theications . Most coFurthermore, some special-purpose resins, that is, heat-resistant or flame-retardant vinyl esters, have been developed using epoxy phenol novolac resins and epoxy resins based on tetrabromobisphenol A, respectively. In conventional, industrially significant systems, monocarboxylic acids, such as acrylic (AA) and methacrylic acid (MAA), serve as a source of reactive unsaturation on the VERs end-group ,3. MoreoTo date, only a few studies have been carried out to develop multifunctional resins comprising both epoxide group and reactive, terminal unsaturation (monoacrylate-terminated epoxy acrylate) ,15,16,172O3 NPs).It should be emphasized that the majority of difunctional EAs were designed to combine the functionalities of both UV- and thermal-curing. In this context, Park Y. et al. investigated the dual curing behavior of composition based on partially acylated DGEBA epoxy oligomers with varying content of a latent curing agent . Su Y. eThe very first commercial multifunctional low viscosity resins were launched by Hexion Inc. under the trade name Epon. The most recognized product in their offer, coded as Epon 8111, is a combination of bisphenol A-based epoxy resin and acrylate . Accordi1H NMR). Subsequently, photoreactive coating compositions were prepared from the obtained methacrylated epoxides and cured using UV radiation. The prepolymers collected during the reaction were subjected to cationic photopolymerization, while the final product was tested for cationic and radical photopolymerization. The samples were tested for the kinetics of the process and the properties of the cured coatings.In this work, the modification of epoxy resin with methacrylic acid, allowing to obtain a polymer containing both acrylic and epoxy groups, has been reported. At first, optimization of modification procedure was followed by a kinetics study, where molar ratio, catalyst dosage and process temperature remained constant and set to optimal conditions, whilst the synthesis time was varied from 60 to 300 min. Pre-polymers properties were studied regarding their chemical structure (FT-IR studies), non-volatile-matter content (NV), partial acid value (PAVs), epoxide equivalent (EE) and viscosity. Moreover, substrates and synthesized epoxy methacrylates were investigated by nuclear magnetic resonance spectroscopy , C-O (~914 cm−1) and C-O-C (~825 cm−1) stretching vibrations of oxirane groups [−1 originating from the carbonyl stretching mode in MAA [−1, thereby suggesting that new bonds occur due to the reaction of carboxyl groups of MAA and epoxide groups of epoxy resin, that is, ester bonds. This is in agreement with the previous studies reporting the shift of the C=O band after the esterification reaction [−1, due to -O-H groups’ evolution [As far as reaction time is concerned, the model synthesis of the epoxy methacrylate (EA) system was performed and characterized in detail in order to establish the impact of the extent of the reaction on the chemical structure, conversion of the substrate, and finally, functional properties of epoxy methacrylate pre-polymers. The samples were collected every hour for a total of 5 h and the progress of the reaction was monitored by FT-IR spectroscopy. The absorption bands corresponding to the vibrations of carboxyl and epoxy groups , partial acid value (PAVs) and epoxy equivalent (EE) were also tracked. Each of these can be regarded as a measure of reaction progress. Thus, in the present study, the relationship between process time and the % of reacted epoxide groups as well as % of reacted MAA was established and expressed as epoxy group conversion (EGC) and methacrylic acid conversion (MAAC), respectively . GeneralThe above results are of the utmost importance because they can guide how to adjust reactions to obtain EAs with desired characteristics. Based on obtained values, one can state that the optimum synthesis time of about 4 h should be adopted to prepare EAs that have both epoxy groups and unsaturated double bonds. In the EA, the epoxy conversion at 240 min is close to 50%, while MAA conversion reaches 89%. Prolonging the reaction time leads to a further increase in the epoxy group conversion up to 52% in 5 h, reducing the number of functional groups capable of cationic polymerization at the same time. 1H NMR spectra also confirm the structure of the obtained epoxy methacrylate. 1H NMR spectra of the dianate epoxy resin, methacrylic acid, and prepared epoxy methacrylate. In both Ep5 and EA spectra, signals about 7.1–7.2 ppm and 6.8–6.9 ppm were observed, attributed to the resonance of aromatic protons, respectively. The signal at 1.63 ppm corresponds to the resonance of methyl protons between the phenyl rings, while the group of signals in the region 2.6 and 3.1–3.3 and 3.8–4.2 corresponds to glycidol protons. In addition, the methacrylic acid and product spectra show signals from the vinyl group at 6.10/5.64, and 6.21/5.84, respectively.The 13C NMR spectrum shows that carbon resonance accords well with the predicted molecular structure : δ in ppm 7.16 , 6.87 , 6.12 , 5.64 , 4.26–4.30 , 4.07 , 3.97 , 3.85 , 3.60–3.71 , 3.34–3.25 , 2.94 , 2.83 , 2.71 , 2.27 , 1.92 , 1.63 ; 13C NMR  δ in ppm: 166.58, 156.63, 143.32, 136.43, 127.63, 127.61, 127.56, 125.03, 113.90, 69.16, 67.79, 67.68, 65.72, 49.77, 43.56, 41.37, 30.51, 29.76, 17.57.In a first approach, the photoinitiated cationic polymerization of the pre-polymers collected throughout the reaction was monitored through the photo-DSC method. Photo-DSC offers a simple method of characterizing the UV-curing kinetics for the photopolymerization of UV-cured materials. Therefore, the profiles for the heat of reaction versus time provided by photo-DSC can be used to describe the photoinduced reaction kinetics and evaluate the polymerization rate. The compositions used in the study included reaction mixtures obtained at one-hour intervals and a cationic photoinitiator (4 wt%). In the plot, the UV irradiation was initiated the reaction mixtures with a cationic photoinitiator, and the photopolymerization reaction occurred immediately in the presence of UV light. The total area under the thermogram curves was the highest for samples with lower viscosity (less than 20 Pa·s). It decreased with the decrease in the concentration of epoxy groups in the reaction mixture. This phenomenon can be reasonably ascribed to the epoxy methacrylate oligomers’ increased molecular weight and conversion degree. As the reaction proceeded, the viscosity of the samples increased significantly, resulting in a decrease in reactivity. Thus, the heat flow decreased due to a decrease in the relative abundance of reactive epoxy groups and viscosity, resulting in a slower reaction rate. In the initial stage of the EA synthesis reaction, there are many unreacted epoxy groups and MAA molecules, hence a double peak is observed, which probably corresponds to the photopolymerization of the epoxy groups of the obtained EA prepolymer as well as unreacted epoxy resin. As the reaction proceeds, the peak corresponding to the photopolymerization of the epoxy groups of the unreacted epoxy resin disappears. As a consequence, a distinct exothermic peak is obtained which corresponds to the actual photopolymerization reaction of the obtained epoxy methacrylate prepolymer.As the reaction progresses and the epoxy groups in the reaction mixture decrease, the tack-free time is extended from 45 to 90 s . Thus, aMoreover, it is worth emphasizing that, owing to the presence of both epoxy and double carbon–carbon bonds in the polymer chain; the resulting EAs are expected to exhibit curing behavior via two distinct mechanisms: (i) cationic ring-opening polymerization; and (ii) free radical polymerization . Therefo−1 (γC-O epoxy), the ether groups at 1036 cm−1, and double bonds at 1635 cm−1 (γC=C methacrylate double bond). −1 exhibit a characteristic absorption band for epoxy groups, the intensity of which gradually decreases with the extension of cationic ring-opening polymerization. Simultaneously, one can observe the increase of characteristic absorption bands for ethers (-C-O-C) at 1036 cm−1 and the presence of methacrylic double bonds at 1635 cm−1. The crosslinking of radical polymerization of EA-R composition was investigated by analyzing the same peaks, in particular the double bonds’ characteristic peak at 1635 cm−1. As the curing time proceeds, the latter diminishes in intensity, suggesting polymerization by unsaturated bonds derived from the methacrylate double bonds. In this case, the presence of epoxy groups is also confirmed after the curing process.IR spectra analyses also observed the cationic and radical crosslinking polymerization of the EA prepolymer. The two investigated coating compositions  exhibit different IR spectra after the curing process. The spectra differences of uncured and cured EA samples can be observed by revealing a presence of characteristic absorption bands for epoxy ring stretching vibration (-C-O), ether groups (-C-O-C), and double bonds derived from the methacrylic groups (C=C). The presence of epoxy groups in IR spectra was proved from the presence of strong bands at 915 cm−1 due to C-O stretching in the epoxy ring. The peak of the epoxy group at 915 cm−1 decreases during the UV curing process, as shown in the IR spectra. Simultaneously, the extent of double bond reactions of EA-R was determined by the peak areas of the double bond peak at 1635 cm−1.Kinetics of photopolymerization is useful for understanding both curing rate and curing degree, particularly in the context of influencing the cationic or radical mechanism of the photopolymerization process. The epoxide and unsaturated double bond conversion (DC), as well as photopolymerization rate (Rp) versus irradiation time, were investigated employing the FT-IR method . As expe−1 (reactivity understood as the loss of epoxide groups over time). Other results have been obtained by using a radical photoinitiator. In this case, the double bond conversion was about 50 % after the same time as the cationic process, and the polymerization rate was found to be 40 s−1. It is well-known that during the reaction of the UV-curing system, the polymer’s mobility and the diffusion capacity of the active radicals greatly influence the conversion efficiency [The cationic photopolymerization proceeds slowly, probably due to severe mobility restrictions in the solid uncured film and reaches only 32 % conversion after 60 min UV exposure. From the initial slope of the recorded polymerization profile, the formulation reactivity was evaluated as 0.7 sficiency . Therefo® (Ep5); with epoxide number of 0.48–0.51 mol/100g and viscosity ranging from 20,000 to 30,000 mPa·s at 25 °C) were purchased from “Organika-Sarzyna” S.A., Nowa Sarzyna, Poland. Hence, the following materials were used to synthesize hybrid oligomer: methacrylic acid (MAA), stabilized, with the purity of 99.5%, was supplied by Acros Organics, Geel, Belgium. Triphenylphosphine (PPh3), Apollo Scientific, Bredbury, UK, was used as a catalyst in the reaction between Ep and MAA, while hydroquinone  was used as a polymerization inhibitor. All chemicals were employed as received. The epoxy resin  and crystal violet purchased from Chempur ; chloroform form P.P.H. Stanlab ; ethyl alcohol from Avantor , tetraethylammonium bromide provided by Acros Organics , perchloric acid standard solution 0.1 M in glacial acetic acid supplied by Fischer Chemicals , Phenolophtalein 1% in ethyl alcohol solution from Eurochem BGD . All chemicals were analytical grade and were used as received. Bis(dodecylphenyl)iodoniumhexaflouroantimonate in propylene carbonate  was applied as a cationic photoinitiator and ethyl-phenyl phosphinate  was applied as a radical photoinitiator. In addition, glycidyl methacrylate was used as a reactive diluent.The synthesis of epoxy methacrylate (EA) resin was carried out in a 250 mL three-neck glass reactor , into which bisphenol A type epoxy resin and hydroquinone as a radical scavenger  was added into the reactor at room temperature. Then, methacrylic acid  and catalyst–triphenylphosphine in the amount of 0.8 wt% (relative to the mass of MAA) were added. The mixture was heated to 70 °C using an oil bath and stirred 120 RPM) to obtain the homogenous mixture. The final reaction temperature was set to 90 °C, and the process was conducted for 5 h in a nitrogen atmosphere, with stirring. The above synthesis parameters were established based on a literature survey  RPM to o,22,23 an−1, at room temperature.The infrared spectra were acquired with a Thermo Nicolet 380 FT-IR spectrometer. Sixteen scans were averaged for each sample in the range of 4000–400 cm1H NMR and 13C NMR. The 1H and 13C NMR spectra were recorded in d6-acetone containing 0.03% (v/v) TMS (tetramethylsilane) on a BRUKER DPX-400 Avance III HD spectrometer  operating at 400.13 MHz (1H) and 100.62 MHz (13C).The prepared epoxy methacrylates were identified by 2/m1) × 100%, where m1 is the weight of the EA sample; m2 is the residual weight of the sample after heating.The non-volatile-matter content (NV) was evaluated thermogravimetrically, using moisture analyzer MAX 60/NP , according to ISO 3251:2019 standard. The analysis was performed at 140 °C for 30 min. The sample weight was approximately 1 g. NV (%) =  were determined by colorimetric titration according to EN ISO 2114:2000 standard, following the procedure described in detail elsewhere . s0 is the initial value of PAVs (mg KOH/g).The PAVs values were used to estimate methacrylic acid conversion (MAAC) according to the following equation: Epoxy equivalent (EE) was determined by means of colorimetric titration, according to EN ISO 3001:1999 standard following the procedure reported in our previous work [0 is the initial value of EE (g/mol); EEmeasured–EE value at a certain time.Epoxy group conversion (EGC) was estimated from EE measurements via the following formula: The viscosity tests were carried out using a cone-plate viscometer LAMY RM-100 plus CP 2000 at room temperature.2 to dryness.The coating compositions have been formulated using synthesized epoxy methacrylates and 4 wt% PI . The components were stirred together under dark conditions until a homogeneous mixture was obtained. Subsequently, the curing solution was applied to the glass substrates employing a gap applicator (120 µm). The polymeric film was cured under a light source  at room temperature and irradiated under UV light with an intensity of 200 mW/cm2, Excelitas Technologies, Mississauga, ON, Canada). As a result, a polymerization solution was composed of epoxy methacrylate resin and 4 wt% of photoinitiator .The UV-curing process of epoxy methacrylate varnishes was isothermally monitored (25 °C) for 15 min by means of photo-DSC apparatus  equipped with UV light emitter Omnicure S2000  was used to determine the conversion of epoxide groups of methacrylic double bonds. More importantly, this spectroscopic technique permits in situ monitoring of the chemical processes via mimicking the disappearance of the characteristic bands of the reactive monomer subjected to UV exposure [2) and an IR analysis light beam. The absorbance change of the epoxide group (C-O) and methacrylate double bond (C=C) peak area was correlated to the extent of polymerization. The degree of conversion (DC), can be expressed by the following relations: DC (%) = (A0-At) · 100/A0, where A0 is the initial peak area before irradiation and At is the peak area at time t. The photopolymerization rate (Rp) was calculated by the following relations: Rp = dDC/dt, where t is the time of irradiation [Fourier transform infrared spectra (FT-IR) were obtained on a Nicolet iS5 instrument. The resolution is 4 cmexposure . The EA adiation .The following tests were performed in order to evaluate the properties of cured coatings: tack-free time, pendulum hardness test, gloss and yellowness index. Tack-free time was measured as a surface cure time according to ISO 9117. It is the time at which the coating is deemed to be properly adhered to and achieves the final technical parameters. The hardness of coatings was tested using Persoz pendulum hardness on the glass substrate  according to ISO 1522 standard. Gloss was measured by spectrometer GLS at an angle of 20 degrees  according to ASTM D523. Yellowness Index is a number calculated from spectrophotometric data that describes the change in the color of test samples. This parameter was measured according to ASTM E313 using precision colorimeter NH-145 .In this paper, the synthesis of epoxy methacrylate resin (EA) by modification of epoxy resin with methacrylic acid has been shown. Moreover, coatings preparation, based on synthesized resin and using UV radiation has been presented. The obtained EA prepolymer, which contains both epoxy and vinyl groups in their structure , made it possible to produce coatings by two different photopolymerization mechanisms-cationic or radical. The optimization of the modification procedure, as well as the photo-crosslinking process, was followed by a kinetics study. The photoinitiated cationic polymerization of the prepolymers was monitored by means of the photo-DSC and FT-IR methods. Heat flow peaks related to the reaction kinetics of EAs prepolymers decreased as the reaction progresses, which is due to a decrease in the number of epoxy groups in the prepolymer chain at the expense of adding methacrylic groups. It also obtained cured coatings with a single exothermic peak, proving the photopolymerization of photoreactive groups in the obtained epoxy acrylate prepolymer. The properties of the cured coatings were characterized. Moreover, it has been shown that the radical process leads to similar properties of cured coatings as the cationic process does, which can be obtained in a shorter time.


Wnt5a is one of the potent signaling molecules that initiates responses involved in cancer through activation of both canonical and noncanonical signaling cascades. Wnt5a both directly and indirectly triggers cancer-associated signaling pathways based on the cancer type. In colorectal cancer (CRC), altering Wnt5a expression can influence several cellular processes of tumor cells, including proliferation, differentiation, migration, invasion, and metastasis. This review summarizes the molecular mechanisms and clinical importance of Wnt5a in the pathogenesis of CRC for better understanding the pathogenesis and its potential role as a prognostic marker and as an appropriate therapeutic target in the treatment of this disease in the future. Wnt cascades , 4.β-catenin signaling, the expression of target genes is regulated by the presence and absence of Wnt ligands [β-catenin is degraded and ubiquitinated by key components of the destruction complex, including glycogen synthase kinase 3β (GSK3β), adenomatous polyposis coil (APC), and Axin [β-catenin into the nucleus where it tightly binds to specific transcription factors such as T cell factor/lymphoid enhancer-binding factor (TCF/LEF) to stimulate expression of target genes involved in cell proliferation and differentiation [In canonical Wnt  ligands , 6. In tand Axin , 8. Follntiation , 10.β-catenin signaling, noncanonical Wnt signaling, independent of β-catenin stabilization, regulates expression of Wnt noncanonical ligand and several cellular processes, including through activation of both Wnt/Ca2+ and planar cell polarity (PCP) [+ signaling is also triggered by the interaction between noncanonical Wnt ligands such as Wnt5a and the Ror2 receptor, which leads to the activation of phospholipase C- (PLC-) induced inositol 1,4,5 triphosphate (IP3), and 1,2 diacylglycerols (DAG) production forms membrane-bound phosphatidyl 4,5-bisphosphate [κB (NF-κB) and protein phosphatase calcineurin- (Cn-) induced nuclear factor associated with T cells (NFAT) [κB [In contrast to the canonical Wnt ty (PCP) , 12. Folty (PCP) . Regardity (PCP) , 15. Folty (PCP) , 17. In hosphate . Then, as (NFAT) , 20. EveFAT) [κB .β-catenin is also activated by Wnt5a [β-catenin signaling through translocation of β-catenin into the nucleus overexpression Fzd-4 and low-expression of Ror-2 in orthodontic tooth movement (OTM) [β-catenin signaling, it has been shown that Wnt5a promotes cyclin D1 through transactivation of ErbB1 and provoking the β-catenin/TCF pathway in Wnt-expressing HC11 mammary cells [β-catenin signaling, Wnt5a is also able to inhibit canonical Wnt signaling via binding to the specific receptors, β-catenin degradation, and loss of Wnt β-catenin target genes expression [β, can also inhibit canonical signaling pathways by β-catenin degradation and low-expression of Wnt3a in HEK 293 cell lines [β-catenin signaling through binding to the Ror2 receptor, downregulation of β-catenin, and reducing Wnt3a-mediated SuperTOPFlash (STF) luciferase activation has been proved in a dose-dependent manner of Wnt3a in 293 cells [According to the reciprocal relationship between noncanonical signaling and expression of noncanonical Wnt proteins, Wnt5a also activates noncanonical signaling cascades via binding to several receptors, including receptor tyrosine kinase-like orphan receptor 2 (Ror2) and Fzd receptors such as Fzd-4 . Like noby Wnt5a . In addiby Wnt5a , 25. In nt (OTM) . In furtry cells . In contpression , 29. In ll lines . In a st93 cells .β, Axin, CK1α, APC, and β-catenin, are effectively considered as an oncogene and tumor suppressor which could be an appropriate therapeutic target in cancers [β-catenin signaling or canonical Wnt signaling is Wnt5a [Consistent with these results, there are generally several factors involved in dysregulation of cellular processes-induced initiation to cancer progression, including genetic and epigenetic alters, which led to activating canonical and noncanonical Wnt signaling aberrantly . In line cancers . One of is Wnt5a . Recent is Wnt5a , 34.Tumor suppressor and oncogenic effects of Wnt5a noncanonical ligands have been shown to be mediated by targeting processes related to cellular senescence, cancer stem cells, chemotherapy resistance, tumor microenvironment cells, cancer-associated inflammation, epithelial-mesenchymal transition (EMT), metastasis, cell proliferation, cell invasion, and cell migration in a wide range of various cancer cell lines . MoreoveThe presence of epigenetic changes of Wnt5a, including methylation, in different stages of some cancers such as colorectal cancer (CRC) has shown that aberrant methylation levels of Wnt5a are not significantly correlated with clinicopathological characteristic including maximal tumor size, tumor extent, tumor site, histology, and Dukes stage . In contβ-catenin protein levels and inactivation of Wnt β-catenin signaling [β- (TGFβ-) treated SW480 cells [Based on the findings of recent studies and the available evidence on the suppressive effect of Wnt5a on types of cell lines and tumor tissue of CRC, this protein can be considered a key therapeutic target in inhibition of CRC progression . The epiignaling . To deteignaling . Regardiignaling . The relignaling . In anotignaling . In furtignaling . Since t80 cells .β-catenin signaling [β-catenin signaling [α (TNF-α) and CaSR. Following CaSR activation, expression of Wnt5a is significantly increased, which leads to the reduction of TNF receptor 1 (TNFR1) and TNF-α-stimulated Wnt3a canonical signaling expression through overexpression of Ror2 in HT-29 CRC cell lines [β signaling [β-catenin and Wnt β-catenin target genes involved in cell proliferation. Conversely, following transfection of pcDNA-Wnt5a and XAV-939 inhibitor into the SW480 cells suppresses cell viability, migration, and invasion of SW480 cells via inhibition of miR-567-5p activity and inactivation of Wnt β-catenin signaling, respectively [In addition to the loss of Wnt5a expression-determining epigenetic modifications factor, Wnt5a-stimulates overexpression of tumor suppressor genes. The factors involved in the overexpression of Wnt5a also demonstrate the inhibitory function of Wnt5a in CRC cells. In line with this, treatment of CRC cell lines including HT-29 and Caco-2 with recombinant Wnt5a (rWnt5a) or Foxy-5, a Wnt5a mimicking peptide, shows that this recombinant can inhibit cell proliferation and stimulate cell differentiation through overexpression of a tumor suppressor enzyme in prostaglandin E2 catabolism named 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and reduction of Wnt ignaling . To stimignaling . Since tll lines . Regardiignaling . In furtectively . Like miIn several recent studies, the association between the loss of Wnt5a expression and clinicopathological features in the different stages of CRC has been examined. Consistent with the histone modification-induced hypermethylation of Wnt5a, it has been determined that downregulation of Wnt5a has a positive correlation with advanced TNM stages of III and IV as well as lymph node invasion in CRC tissues . On the In contrast to the inhibitory effect of Wnt5a-determining factors, including epigenetic modification-induced methylation of Wnt5a promoter, factors-stimulated overexpression of Wnt5a, and Wnt5a-induced overexpression of tumor suppressor genes, the stimulatory effect of Wnt5a on CRC samples has also been examined in several studies. Regarding the oncogenic effect of Wnt5a, this protein can potentially be influenced on CRC cells mediated by specific cells, including tumor-associated macrophages (TAMs). In line with this, findings of a study showed that Wnt5a expressed by TAMs stimulate tumor cell proliferation and migration of CRC cells, including HCT116 and DLD1, and recruited macrophage infiltration through enhancing the CaMKǀǀ-ERK signaling pathway . Accordi2+ channel. The TrpC5 is highly expressed in CRC cells; this permeable Ca2+ channel can play a crucial role in regulating the differentiation of colon tumor cells. It has been demonstrated that TrcP5-expressing poorly differentiated CRC cell lines including SW480, HT-29, SW620, DLD1, HCE8693, and RKO can upregulate Wnt5a and cytoplasmic β-catenin expression that finally lead to the decreasing differentiation and enhancing stemness of CRC cells [γ), which is known as a downstream target of Wnt5a noncanonical signaling induced by dextran sodium sulfate- (DSS-) induced colitis-associated cancer (CAC). It has been observed that DSS promotes inflammatory signals and injuring IECs via overexpression of Wnt5a noncanonical ligand and receptors and coreceptors of Wnt5a, including Fzd-2, -5, and Ror2. Following overexpressed Wnt5a and its receptor/coreceptor in IECs, Wnt5a significantly increases the phosphorylation of CAMK2γ, which eventually leads to the stimulation of survival and proliferation of colonic epithelial cells through overactivation of epithelial signal transducer and activator of transcription 3 (STAT3) signaling during CAC development [Consistent with the regulatory and secretory factors in the tumor microenvironment region, a member of the transient receptor potential channel (TrpC) family named TrpC5 forms a receptor to activate the nonselective Caelopment . Anotherelopment .This review summarizes the recent findings on the clinical importance and function of Wnt5a in the pathogenesis of colorectal cancer (CRC) Tables  and 2. T


Benign phyllodes tumor (BPT) and fibroadenoma (FA) have some difficulties in differential diagnosis. BPT is often misdiagnosed as FA during the first operation and is not diagnosed until postoperative recurrence and reoperation. The intent of this research was to find and validate microRNAs (miRNAs) with significant differential expression between BPT and FA as novel potential differential biomarkers.P value < 0.05 were considered statistically significant.Tissue specimens from three BPT patients and three FA patients were selected to detect the expression of miRNAs by miRNA-Seq technique. Primary cells were extracted and cultured from fresh BPT and FA tissues by tissue-block culture. The expression of differentially expressed miRNA (DEmiRNA) was further verified by quantitative real-time polymerase chain reaction (qRT-PCR) in twelve BPT and eleven FA patient specimens as well as primary cells. Data with a The miRNA-Seq results showed totally six DEmiRNA were identified, consisting of two downregulated genes and four upregulated genes in BPT. Further validation by qRT-PCR manifest that miR-140-3p was downregulated by approximately 70% in BPT.miR-140-3p could become potential differential biomarker for BPT and FA.The online version contains supplementary material available at 10.1186/s12905-022-01613-4. Phyllodes Tumor (PT) accounts for about 0.3–1% in all primary breast tumors . UsuallyBorderline PT and malignant PT are clearly distinguished from FA, while BPT and FA have similar clinical, imaging, and cytopathological findings . ExistinMicroRNAs (miRNAs) are an evolutionarily conserved class of endogenous single-stranded non-coding small RNAs of approximately 22 nucleotides in length that account for approximately 3% of the human whole genome . miRNAs Several published articles have reported that miR-140-3p plays an important part in a variety of diseases. miR-140-3p can act as potential biomarker for osteoarthritis, autism spectrum disorder, osteoporosis and osteoporotic fractures –20. In cThe written informed consent of each patient participating in the study was obtained. The study protocol and informed consent were approved by the Ethical Committee of Qilu Hospital of Shandong University. All of the methods were carried out in accordance with the Declaration of Helsinki.All 25 specimens required for this study were obtained from patients admitted to the Department of Breast Surgery, Qilu Hospital of Shandong University for surgical treatment from January 2017 to January 2021. After surgery, all of the tumors were subjected to a histopathologic diagnosis by two pathologists. Due to the small sample size, we only grouped the samples by pathology. All tissues used in the experiments were stored in liquid nitrogen after surgical resection.Briefly, an appropriate amount of tissue was cut from the specimen and 1 ml of Trizol  was added and ground in a grinder until flocculent. The ground tissue was subjected to subsequent procedures according to the trizol method. The concentration of RNA was measured and adjusted to 200–500 ng/μL for subsequent manipulation.The Human miRNA-seq was performed by KangChen Biosciences (China). Detailed steps are provided in Additional file 2O and heated at 42 degrees for 2 min to remove gDNA. Then, reagents 3 and 4 and reverse transcription primers were added, and dd H2O was added to make the total liquid volume of each reaction system reach 20 μL. Set the heating conditions to 42 degrees for 15 min and 85 degrees for 5 s. The resulting cDNA was subjected to PCR in a Step One real-time PCR machine  following the instructions of the FastStart Essential DNA Green Master , and a melting curve was drawn. The final concentration of PCR primers was 0.3 μM. miR-10b-5p primers  for PCR were: 5′‐GGCGAAGAGTACCCTGTAGAACC‐3′ (sense) and 5′‐TATGGTTGTTCACGACTCCTTCAC‐3′ (antisense). miR-126-5p primers  were 5′‐TGGTGGAGGCATTATTACTTTTGG‐3′ (sense) and 5′‐GTGCAGGGTCCGAGGT‐3′ (antisense). miR-140-3p primers  for PCR were: 5′‐TCACTGCTCTGTACCACAGGGTAG‐3′ (sense) and 5′‐ TATGGTTTTGACGACTGTGTGAT‐3′ (antisense). U6 primers  were 5′‐CAGCACATATACTAAAATTGGAACG‐3′ (sense) and 5′‐ACGAATTTGCGTGTCATCC‐3′ (antisense). Relative expression level was computed using 2−ΔΔCt method.One thousand ng of total RNA from each sample were reverse transcribed by PrimeScriptTMRT regent Kit with gDNA eraser  with a final reverse transcription primer concentration of 0.25 μM. First, reagents 1 and 2 were added to each reaction system according to the instructions, and the total amount of liquid was supplemented to 10 μL by dd HPrimary cells were extracted by tissue-block culture method: small pieces of fresh tissue were cut and washed with sterile PBS for 3 times; surface fat and fascia were removed, cut to small pieces with a diameter of about 1 mm, and spread flat to the bottom of culture flask, with the distance between small pieces of tissue being about 0.3 cm; the flask was turned upwards, an appropriate amount of culture medium was added, and the culture flask was placed in a constant temperature incubator . After cultured for 2–4 h, the flask was slowly turned over after the small pieces dried up. Cell sprouting can be observed after 24 h.P value < 0.05 in the statistics were considered statistically significant.All data from this experiment were analyzed by GraphPad Prism 7 . For each sample, RNU6 was used as an internal reference to calculate the Δct value of each miRNA separately. The Δct values of each miRNA in the FA group were averaged and used as the control group to calculate the ΔΔct values of each sample, respectively. Finally, the relative expression level of each miRNA for each sample was obtained by calculation ) and analyzed with this data. Expression levels of each sample from different groups were statistically analyzed by unpaired t-test, and the data with The experimental design of this study is shown in Fig. Primary cells were extracted by tissue-block culture from two FA and two BPT tissues Fig. D, E, andmiRNAs play an important role in tumor development by regulating the expression and activity of downstream target genes or other signaling molecules. Upregulated or down-regulated miRNAs may result in the suppression of the expression of oncogenes or tumor suppressor genes, suggesting that miRNAs may serve as both tumor suppressor genes and oncogenes . In receIt is important to find miRNAs as potential differential diagnostic biomarkers for BPT and FA. In this study, we screened six miRNAs with fold changes greater than 1.2 with statistically significant among 695 miRNAs by miRNA-Seq technology, of which two miRNAs were down regulated (miR-140-3p and miR-126-5p) and four miRNAs were up regulated  and miR-novel-chr233028) in BPT. By observing the tag count, three miRNAs with higher expression content were selected for validation. Compared with FA patients, the expression level of miR-140-3p in tissues from BPT patients was significant decrease. It can be found by scatter plot that the expression within the BPT group was stable at a lower level, while the miR-140-3p expression level within the FA group fluctuated greatly, but almost all were higher than those in the BPT group were. We believe that due to some similarity between FA and BPT in organization, the miR-140-3p expression level in some FA is close to BPT, but the level of miR-140-3p in most FA is still higher than BPT. In validation set, the difference trend of miR-126-5p and 10b-5p between BPT and FA groups was the same as that of the screening group, but the expression levels of them in tissues were unstable, and it was difficult to determine a boundary to distinguish the two diseases. Due to the lack of a sufficient number of BPT specimens, intragroup differences are reduced by expanding the sample size or setting grouping conditions. Meanwhile, primary cells were extracted from 2 FA and 2 BPT tissues, respectively, and the expression levels of miR-140-3p were detected, and it was found that cells also showed the same difference situation as tissues. Both screening and validation results showed that the expression level of miR-140-3p was significantly different in FA and BPT, while the differential expression was the same between cells and tissues, and the expression level of miR-140-3p was stable in BPT, so we concluded that miR-140-3p could serve as a potential biomarker to differentiate FA from BPT.In order to establish a miRNA as a biomarker for differentiating FA from BPT in clinic, the following issues need to be solved. First, to find a miRNA with significant expression differences between FA and BPT, this study demonstrated that the expression of miR-140-3p is significantly different between FA and BPT through screening and validation, and the potential mechanism of the effect of this miRNA on the development of BPT still needs further study. Second, from the validation results, it can be seen that the expression of miR-140-3p is more stable in tumor tissues of BPT patients, but the expression fluctuates greatly in FA patients, and it is still necessary to expand the sample size to further verify the feasibility of miR-140-3p as a molecular biomarker. Finally, if miR-140-3p serve as a clinical differential index, a boundary or interval needs to be determined to distinguish the two diseases, FA and BPT, which has higher requirements for specimen volume as well as detection methods. When performing preoperative needle biopsy and intraoperative rapid frozen pathology, a marker with differential significance helps physicians make a correct diagnosis and differential diagnosis of the patient's condition, resulting in better treatment.In summary, this study found and validated significant differences in the expression levels of miR-140-3p between FA and BPT patient tissues by screening. Compared with FA, miR-140-3p expression levels were down-regulated by about 70% in BPT patient tissues, and miR-140-3p expression levels were also decreased in BPT primary cells, and we concluded that miR-140-3p can be used as a potential molecular biomarker to differentiate FA from BPT, while miR-140-3p may regulate the development of BPT by targeting one or more target genes. The experimental results of this study have some guiding significance for studying the occurrence and development of FA and BPT.Additional file 1. Methods of Human miRNA-seq and Date analysis.