According to mass spectrometry, the phosphazene fractions of PERO include up to 30 individual compounds with a predominance of cyclotriphosphazenes with one unsubstituted chlorine atom and four or five glycidyl groups. PERO has a lower viscosity in comparison with similar resins based on bisphenol A, which can simplify their use as a binder for polymer composites, adhesives, and paints.Phosphazene-containing epoxy-resorcinol oligomers (PERO) are synthesized in one stage with the direct interaction of hexachlorocyclotriphosphazene (HCP), resorcinol, and epichlorohydrin in the presence of solid NaOH. Depending on the initial ratio of HCP:resorcinol, PERO contains from 20 to 50 wt.% phosphazene component (2.0–4.8% of phosphorus) and have an epoxy group content up to 30 %. Products are characterized using  Epoxy resins have won a special place in industry and everyday life—though not so much in terms of production, but in their specific role . ClassicA rather effective way to increase the heat resistance of epoxy matrices is their structural modification, in particular, with compatible oligomers with higher functionality, which can be incorporated into the three-dimensional network structure formed during curing ,4. ReducOne of the promising classes of compounds that can be used as modifiers that reduce the flammability of polymers are phosphazenes ,12,13. TIn order to avoid possible negative effects arising from the use of additive type flame retardants, it is preferable to ensure the formation of covalent bonds of the flame-retardant molecules with the matrix of the modified polymer. Therefore, halogenphosphazenes, of course, are of interest as a universal, flexible building block, which makes it possible to easily obtain functional organophosphazenes as well.Phosphazenes with reactive functional groups in an organic substituent, when introduced into thermosetting matrices, perform not only the function of a flame retardant, but can also change the structure of the three-dimensional network and positively affect the performance properties of the polymer. Thus, functional phosphazenes increase fire resistance , thermalEpoxy resins modified with epoxyphosphazene are characterized by low flammability, increased heat resistance and char yield, hydrophobicity, and non-toxicity of combustion products ,29,30,31Currently, there are 2 main synthetic approaches that allow obtaining functional phosphazenes capable of forming covalent bonds with epoxy matrices: synthesis of organophosphazenes with reactive epoxy groups ,53,54,55In Kireev et al.’s study , severalThe simplest, most convenient and promising method for obtaining PEO—based on hexachlorocyclotriphosphazene (HCP) and bisphenol A—is a one-step one-pot process of the interaction of these monomers in the presence of an alkaline agent in an environment of excess epichlorohydrin as a reagent and solvent ,70.Most of the above-described phosphazene epoxides are solids or highly viscous liquids that can reduce the processing properties of modified epoxy resins and make it impossible to process them at room temperature.Thus, the development of new simple methods for obtaining of aryloxyphosphazene-containing epoxy resins—based on readily available, inexpensive starting reagents with reduced viscosity and improved processed properties—is of great scientific and practical interest and may contribute to accelerating the widespread use of phosphazene-containing resins as components of high-tech polymer composite materials.In order to obtain a low viscosity PEO with greater phosphorus content in the present work, instead of bisphenol A, resorcinol was used, the interaction of which with HCP and epichlorohydrin was performed according to the scheme in 31P-singlet spectrum with δP = 19.9 ppm, was obtained by the method [Hexachlorocyclophosphazene—a white crystalline substance with m.p. of 113 °C; NMR e method .99.5% pure  sodium hydroxide (NaOH) from JSC KAUSTIK , in white granules with a diameter of 1–2 mm was used without purification. The content of crystallization water determined by acid-base titration was about 5%.Epichlorohydrin (Solvay) with the content of the main substance of 99.8% was distilled before use, b.p. 118 °C.Solvents were purified according to known methods, their physical characteristics corresponded to the literature data .The viscosity of the PERO obtained was compared with D.E.R. 332 epoxy resin  with the epoxy group content of 24.6–25.1% and epoxy equivalent of 171–175.In order to adjust the content of the phosphazene component in the product, a series of experiments was carried out according to the methods below, changing the amounts of the initial reagents in accordance with the In a four-necked flask with a volume of 250 mL, equipped with an overhead bladed stirrer, distilling trap, and thermometer, 1.00 g (2.87 mmol) HCP, 5.06 g resorcinol (46.0 mmol) and 100 mL epichlorohydrin were charged. The mixture was heated in an oil bath until the reagents were completely dissolved and the epichlorohydrin boiling point was reached. Then, with vigorous stirring, 3.86 g (96.6 mmol) of granulated sodium hydroxide was immediately charged. The reaction was conducted for 30 min, maintaining the boiling of the reaction mixture and distilling off the azeotropic mixture of epichlorohydrin with evolving water. Then the flask was cooled to room temperature, and the epichlorohydrin was distilled off in vacuum at a temperature not higher than 90 °C. The contents of the flask were extracted with 200 mL of acetone, and the formed sodium chloride and possible excess alkali were filtered off. The precipitate was extracted with another 200 mL of acetone, the solvent was distilled off from the combined solutions, and the residue was dried in vacuum at 90 °C until constant weight. 7.04 g (69%) of a viscous transparent light-yellow product with epoxy group content of 22.2% was obtained.Synthesis of resorcinol-based phosphazene-containing epoxy resin with the gradual loading of alkali was carried out as above and with the indicated amounts of the initial reagents, with the exception that all the calculated amount of alkali was divided into 6–10 equal portions of approximately 0.5 g and added with an interval of 3 min after which the reaction was carried out for 30 min, maintaining the boiling of the reaction mixture and distilling off the azeotropic mixture of epichlorohydrin with evolving water. The product was isolated in the same manner as above.9.08 g (89%) of a viscous transparent light-yellow product with epoxy group content of 28.6% was obtained.31P and 1H NMR spectra were measured in chloroform-d solutions with a Bruker AV-400 spectrometer  operating at 162 and 400 MHz, respectively. The signals due to the deuterated solvents were used as internal references. The chemical shifts of the signals were calculated relative to the signals of tetramethylsilane (1H) and phosphoric acid (31P), which were used as references. The spectra were processed with the help of the MestReNova Lab software package .The MALDI-TOF mass spectrometric analysis was carried out on the Bruker Auto Flex II instrument .Elemental analysis was carried out by spectrophotometry.2O) divided by the total molecular weight of epoxy resin. Thus, the theoretical content of epoxy groups was calculated by the formula:M are the molecular weights of the oxirane group and the whole epoxy resin molecule, respectively, and n is the number of oxirane groups in the epoxy resin molecule. The experimental epoxy group content was determined by the method of reverse acid-base titration [V is the amount of NaOH solution for titration of a sample containing epoxy resin in mL; V0 is the amount of NaOH solution for titration of blank sample without epoxy resin in mL; 43 is the molecular weight of the oxirane group; N is the concentration of NaOH solution, mol∙L−1; and g is the mass of epoxy resin sample in g.In the present paper, epoxy group content (in wt.%) is the weight of oxirane groups , it is necessary to use diphenol in an amount that provides at least a twofold excess of OH-groups relative to P–Cl bonds [Previously, using the example of a bisphenol-A based PEO, it was shown that to eliminate gelation in highly functional ACl bonds ,70.From 31P spectra  and 4.2 ppm (tt)) and penta-substituted  and 6.2 (d)) trimeric cycles. The low-intensity singlet signal at 7.9 ppm in the 31P NMR spectra indicates an insignificant content of hexa-substituted triphosphazene compounds in PERO.As in the case of bisphenol A based epoxyphosphazene oligomers ,70, the  spectra  of signa31P and 1H spectra of the reaction mixture do not change.Determination of the optimal temperature and duration of the process was carried out at the HCP:resorcinol ratio of 1:16, at which the calculated phosphorus content in the product is 3.0% and an acceptable level of fire resistance is reached. Experiment number 2 was carried out in accordance with the terms of the patent : the rea1H NMR spectra of the PERO  when charging of all the reagents into the system at once, the PERO yield ranges from 55–57% , while tthe PERO  confirmsThe quantitative content of CG and DH groups in the PERO composition can be estimated from the MALDI-TOF mass spectra , the ass1H NMR spectra, the content of 2-hydroxypropylene bridges  in their composition;more complex PERO compositions are formed at the molar ratios of HCP:resorcinol = 1:12, and there is greater presence of compounds whose molecules contain two triphosphazene cycles (m/z = 866) and penta-epoxy (m/z = 996) derivatives with content predominantly being of the latter;in the composition of the phosphazene fractions of oligomers synthesized at the HCP:resorcinol ratio > 1:12, there are mainly tetra- (m/z = 1126) in the composition of PERO;there is an insignificant amount or almost complete absence of hexa-(3-glicidyloxyphenoxy)-cyclotriphosphazene (m/z = 866) and penta-substituted (m/z = 996) compounds are present in the phosphazene fraction.the most homogeneous composition of the phosphazene fraction is PERO synthesized at molar ratios of HCP:resorcinol = 1:16 and 1:24, especially in the case of gradual loading of solid alkali . At a ratio of 1:24, predominantly tetra- , the dehydrochlorination reaction is difficult, as evidenced by the increased content of chlorine and OH groups in the product. This fact is probably due to steric difficulties caused by the increase in the intensity of the gelation reaction. The latter is confirmed by the low yield of soluble oligomers and the high content of compounds with 2 and 3 phosphazene cycles in the soluble part of the product.For the synthesis of PERO, it is preferable to use gradual (portioned) loading of alkali. The most optimal HCP:resorcinol ratio is in the range from 1:24 to 1:16, at which there is achieved: an acceptable phosphorus content of 3 to 5%, epoxy group content is 29-30%, and the minimum content of residual chlorine of 2.2–2.7%.The values of viscosity PERO obtained at a ratio of HCP:resorcinol 1:16 and industrial bisphenol A based epoxy resin  is comparable . At the The synthesis of epoxyphosphazene-containing epoxy oligomers based on hexachlorocyclophosphazene and resorcinol in epichlorohydrin excess media, and with a portioned load of solid NaOH, allows the adjustment of the epoxy group content of the resulting oligomer to within 30% and the phosphorus content from 2 to 5%, by varying the ratio of HCP:diphenol. The viscosity of obtained resorcinol based epoxyphosphazene-containing resins is comparable to conventional bisphenol A based epoxies, and is much lower in comparison to similar epoxyphosphazene resins based on bisphenol A. Thus the obtained epoxyphosphazene resins may be used as a component of a binder for composite materials, adhesives, and paints.


Monanchora , closely related to the northeastern Pacific species Monanchora pulchra, collected from deep waters off the Aleutian Islands of Alaska. Through an assortment of NMR, MS, ECD, computational chemical shifts calculation, and DP4, chemical structures of these metabolites have been characterized as spirocyclic ring-containing sesterterpenoid (1) and cholestane-type steroidal analogues (2 and 3). These compounds exhibited the inhibition of β-catenin response transcription (CRT) through the promotion of β-catenin degradation, which was in part implicated in the antiproliferative activity against two CRT-positive colon cancer cell lines.The Wnt/β-catenin signaling pathway is known to play critical roles in a wide range of cellular processes: cell proliferation, differentiation, migration and embryonic development. Importantly, dysregulation of this pathway is tightly associated with pathogenesis in most human cancers. Therefore, the Wnt/β-catenin pathway has emerged as a promising target in anticancer drug screening programs. In the present study, we have isolated three previously unreported metabolites from an undescribed sponge, a species of  Colorectal cancer is the fourth most prevalent cancer and the third leading cause of cancer-related mortality worldwide. In 2017, approximately 130,000 new cases and 50,000 deaths can be attributed to colorectal cancer in the United States alone c 0.09, CH3OH); 1H and 13C NMR data, see +m/z 479.2407 .Compound (2): colorless gum; [α]c 0.08, CH3OH); 1H and 13C NMR data, see +m/z 407.2926 .Compound (3): colorless gum; [α]c 0.1, CH3OH); 1H and 13C NMR data, see +m/z 451.3187 .Compound  cut-off for the atoms indicated in red  program interfaced in Maestro  with a mixed torsional/low-mode sampling method. Advanced conformational searches were carried out in the MMFF force field, in the gas phase with a 50 kJ/mol energy window and 10,000 maximum iterations based on the original authors’ recommendations . NMR chehttp://www-jmg.ch.cam.ac.uk/tools/nmr/DP4/.The DP4 probability analysis was conducted using an applet available at HEK293 (human embryonic kidney cell), HCT116 (human colon carcinoma cell), SW480 (human colon adenocarcinoma cell), and Wnt3a-secreting L cells were obtained from the American Type Culture Collection . Each cell lines were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and two antibiotics . HEK293-FL reporter (TOPFlash) and control (FOPFlash) cells were constructed and Wnt3a-conditioned medium (Wnt3a-CM) was prepared as reported previously . LuciferThe cytosolic fraction was prepared as previously described . Protein1–3 and further incubated for 48 h. The cell viability from compounds treatment was measured using CellTiter-Glo assay kit  according to the manufacturer’s instructions. All experiments were carried out in triplicate.Cells were dispensed into 96-well plates followed by the treatment with compounds 1) and two new cholestane-type steroids (2 and 3) from a deep-water Alaska sponge. The sponge was taxonomically characterized to be an undescribed species of Monanchora. The chemical structures of these metabolites were elucidated by the interpretation of NMR and MS spectral data, and their absolute configurations were determined by comparison of the experimental and calculated ECD spectra, and computational chemical shifts calculation followed by DP4 analysis. We also demonstrated that the isolated compounds exhibit an antiproliferative effect on two β-catenin response transcription (CRT)-positive colon cancer cell lines, HCT116 and SW480. We further showed that these metabolites inhibit CRT activity by stimulating the degradation of β-catenin. To the best of our knowledge, this is the first report that sesterterpenoid shows inhibitory effects on the Wnt/β-catenin pathway. Among steroidal metabolites, corticosteroids such as glucocorticoid are known to inhibit the Wnt/β-catenin pathway [In summary, we have isolated a new spirocyclic ring-containing sesterterpenoid ( pathway . Collect


The miR-17-92 cluster is involved in animal development and homeostasis, and its dysregulation leads to human diseases such as cancer. In the present study, we investigated the functional link between miR-17-92 cluster and Wnt/β-catenin signaling pathway in ICP2 and DF1 cells. We demonstrated that ectopic expression of either LEF1 or β-catenin increased the promoter activity of the miR-17-92 cluster host gene (MIR17HG) and combined ectopic expression of LEF1 and β-catenin further enhanced the promoter activity; while knockdown of either LEF1 or β-catenin reduced the MIR17HG promoter activity. Both LEF1 and β-catenin could directly bind to the MIR17HG promoter. Furthermore, we demonstrated that low doses of lithium chloride (LiCl), an activator of Wnt/β-catenin signaling pathway, increased MIR17HG promoter activity and the endogenous expression of the miR-17-92 cluster, while high doses of LiCl had the opposite effects. Treatment with XAV-939, an inactivator of the Wnt/β-catenin pathway, reduced the endogenous expression of miR-17-92 cluster. Finally, we found that low doses of LiCl promoted the proliferation of ICP2 and DF1 cells, while high doses of LiCl inhibited the proliferation of ICP2 and DF1 cells. Taken together, our results reveal that MIR17HG is a target of LEF1 and the Wnt/β-catenin pathway and suggest that the miR-17-92 cluster may, at least in part, mediate the proliferation-promoting effect of the Wnt/β-catenin pathway in cell proliferation. MicroRNAs (miRNAs) are single-stranded RNA molecules of 19–22 nucleotides in length, which regulate gene expression post-transcriptionally . They tyMicroRNAs genes are scattered across chromosomes either individually or in clusters, in which two or more miRNAs are transcribed from adjacent miRNA genes within a short distance. The polycistronic miRNA cluster, miR-17-92, is one of the best-studied miRNA clusters. The human miR-17-92 cluster is located in an intron of the miR-17-92 cluster host gene (MIR17HG) on chromosome 13 (13q31.3). The primary transcript of miR-17-92 (pri-miR-17-92) is an approximately 0.8-kb-long polycistron and is sequentially processed to produce seven different mature miR-17-92 cluster members  . PreviouThe transcriptional regulation of the miR-17-92 cluster has been extensively studied over the past decade. The c-Myc oncogene was the first transcription factor shown to directly bind and activate the transcription of MIR17HG . To date. Upon activation of the Wnt/β-catenin pathway, β-catenin enters the nucleus and interacts with transcription factors T-cell and lymphoid enhancer factors (TCF/LEF1) to activate and/or repress transcription of specific target genes  were generated in our lab  with high glucose formulation  supplemented with 10% fetal bovine serum , 100 units/ml penicillin, and 100 μg/ml streptomycin, and incubated at 37°C, 5% CO our lab . ICP2 ceTo activate the Wnt/β-catenin signaling pathway, ICP2 and DF1 cells were treated with lithium chloride (LiCl)  for 24 h in DMEM and DMEM/F12 at concentrations of 3, 15, and 30 mM; sodium chloride (NaCl) was used as a negative control. To inactivate the Wnt/β-catenin signaling pathway, ICP2 and DF1 cells were treated with 10 μM XAV-939  dissolved in DMSO  for 24 h; DMSO was used as a negative control.TM RT reagent Kit with gDNA Eraser   with miRNA-specific stem-loop RT primers  was reverse-transcribed into cDNA using the PrimeScriptTM RT reagent Kit with gDNA Eraser , and the relative mRNA expression was determined as described above. TBP was used for normalization. All primers for stem-loop qRT-PCR and qRT-PCR analyses are shown in Total RNA was isolated from ICP2 and DF1 cells using Trizol reagent . For miRNA expression analysis, the extracted RNA was reverse transcribed using the PrimeScript primers  accordinTo construct a luciferase reporter with the MIR17HG promoter, a 714-bp chicken genomic DNA fragment  was synthesized by GENEWIZ  and cloned into the luciferase reporter vector, pGL3-Basic , to yield the MIR17HG promoter reporter (pGL3-WT-MIR17HG-Luc). The potential TCF/LEF1 binding site of the pGL3-WT-MIR17HG-Luc was mutated from TTTGTT to GAATTC using DNA synthesis, and the resulting reporter construct was designated as pGL3-MT-MIR17HG-Luc. For expression vector constructs, the full-length CDS of LEF1 (NM_205013.2) was amplified from the pooled chicken cDNA using PCR with the forward primer: 5′-(G/A)NNATGNCGCAGCTGCCGGGGGCCGGGG-3′ and the reverse primer: 5′-GATGTAGGCAGCTGTCATTCTGGGG-3′ and cloned into the pEASY-Blunt-M2 vector to generate the LEF1 expression vector (pEASY-Blunt-M2-LEF1). The full-length CDS of β-catenin (NM_205081.1) was amplified using PCR with the forward primer: 5′-CGGAATTCGGGCTGACTTGATGGAGTTGGA-3′ and the reverse primer: 5′-GGGGTACCCAACTGATTACTGTCACCTGG-3′ and cloned into pCMV-HA vector in the EcoR I/Kpn I sites to yield pCMV-HA-WT-β-catenin. In addition, to generate the expression vector for the constitutively active mutant of β-catenin (Δ45-β-catenin), the point mutation was generated from pCMV-HA-WT-β-catenin by using Site-directed Gene Mutagenesis Kit (Beyotime) with the primers 5′-ACGACAACTGCTCCCTTGAGTGGCAAAGGAA-3′ and 5′-TTCCTTTGCCACTCAAGGGAGCAGTTGTCGT-3′, and the Δ45-β-catenin expression vector was designated as pCMV-HA-Mut-β-catenin. All constructs were confirmed by DNA sequencing and successful expression was confirmed by western blotting .5 cells per well in 12-well plates. After 24 h of culture, the cells were transfected with si-LEF1, si-β-catenin, and si-NC, respectively. At 48 h post-transfection, total RNA and protein were extracted from these transfected cells. The knockdown efficiency of LEF1 and β-catenin was tested by qRT-PCR and Western blot analysis.LEF1 siRNA (si-LEF1), β-catenin siRNA (si-β-catenin) and control siRNA (si-NC) were designed and synthesized by GenePharma . The following siRNA sequences were used: si-NC sense (5′–3′) UUCUCCGAACGUGUCACGUTT, si-NC antisense (5′–3′) ACGUGACACGUUCGGAGAATT,si-LEF1 sense (5′–3′) GCUAUCAACCAGAUUCUUGTT, and si-LEF1 antisense (5′–3′) CAAGAAUCUGGUUGAUAGCTT, si-β-catenin sense (5′–3′) GCUUUAGGACUCCACCUUATT,si-β-catenin antisense (5′–3′) UAAGGUGGAGUCCUAAAGCTT. DF1 or ICP2 cells were plated at 1 × 104 cells per well in 48-well plates. After 24 h of culture, the cells were transfected with either 200 ng of Topflash or Fopflash, along with 4 ng of pRL-TK. For MIR17HG promoter activity analysis, ICP2 cells were plated at 5 × 104 cells per well in 24-well plates. After 24 h of culture, the cells were transfected with 500 ng pGL3-WT-MIR17HG-Luc or pGL3-MT-MIR17HG-Luc and 10 ng pRL-TK. For transcriptional regulation analysis, cells were co-transfected with 200 ng pGL3-WT-MIR17HG-Luc or pGL3-MT-MIR17HG-Luc and 200 ng of pEASY-Blunt-M2-LEF1, pCMV-HA-WT-β-catenin, pCMV-HA-Mut-β-catenin, pEASY-Blunt-M2/pCMV-HA, 10 pmol of si-LEF1, si-β-catenin, or si-NC. All cells were transfected using Lipofectamine 2000  following the manufacturer’s protocol. At 48 h after transfection, the cells were harvested and luciferase activity was measured using the dual-luciferase reporter assay system . The luciferase activities were normalized to the corresponding Renilla luciferase activity values.The luciferase reporter plasmids (Topflash and Fopflash) were obtained from the Addgene repository. The Topflash harbors three optimal TCF/LEF1-binding sites, and Fopflash harbors three mutated TCF/LEF1-binding sites. To detect Wnt/β-catenin signaling pathway activity, ICP2 and DF1 cells were plated at 2 × 10Immunofluorescence was performed using Immunofluorescence Application Solutions Kit  according to the manufacturer’s instructions. In brief, Cells cultured on coverslips were treated at the indicated concentrations of LiCl and fixed with 4% (w/v) paraformaldehyde for 15 min. The cells were then washed with PBS and blocked in blocking buffer for 60 min at room temperature. Then cells were incubated with anti-β-catenin antibody  or anti-LEF1 antibody  overnight at 4°C, respectively. The cells were stained with FITC-conjugated anti-rabbit IgG  for 60 min at room temperature, followed by nuclei counter-staining with Hoechst 33342 for 5 min at room temperature . Fluorescent images were examined under a confocal laser microscope .1  were used as a loading control. Immunoreactive bands were detected using the ECL plus detection kit .Nuclear proteins were extracted from ICP2 and DF1 cells treated with LiCl, using the NE-PER nuclear extraction kit . Cells were lysed in RIPA buffer, supplemented with PMSF (RIPA:PMSF = 100:1). Cell lysates were separated by 10% SDS-PAGE and transferred onto nitrocellulose membrane . The blots were incubated with the following primary antibodies: anti-LEF1 , anti-β-catenin , anti-HA , anti-Myc . β-actin  and Lamin BNuclear proteins were extracted from DF1 cells transfected with pEASY-Blunt-M2-LEF1 using the NE-PER nuclear extraction kit . Binding activity of LEF1 to the probes was determined using the LightShift Chemiluminescent EMSA Kit . Biotin-labeled probes harboring the potential TCF/LEF1 binding site and the corresponding mutant probes were synthesized by Genewiz. The sequences of forward and reverse single-stranded probes are shown in Chromatin immunoprecipitation (ChIP) assay was performed using the ChIP assay kit  according to the manufacturer’s instructions. ICP2 cells were transfected with pEASY-Blunt-M2-LEF1, pCMV-HA-WT-β-catenin, or pEASY-Blunt-M2/pCMV-HA. At 48 h after transfection, cells were fixed with 1% formaldehyde for 10 min at room temperature and quenched with 125 mM glycine for 5 min at room temperature. The DNA was digested with 0.5 μl micrococcal nuclease into an average fragment size of 100–900 bp. The sample was incubated with anti-β-catenin , anti-LEF1 , or the normal rabbit IgG  as a control. The immunoprecipitates were collected, washed, eluted, and then the cross-links were reversed. The DNA was purified and analyzed by qPCR using the site-specific PCR primers . Non-imm4 cells/well. After 12 h, the cells were treated with different LiCl concentrations  for 0, 24, 48, 72, and 96 h. The control cells were treated with NaCl . At designated time points, 20 μL of CCK-8 solution was added to each well of the plates, followed by 1.5 h incubation at 37°C. The absorbance was measured at 450 nm. All experiments were repeated at least three times.Cell proliferation was determined using the Cell Counting Kit-8 (CCK-8)  according to the manufacturer’s instructions. Briefly, ICP2 and DF1 cells were seeded onto 48-well plates at 1 × 10t-test using GraphPad Prism Software. Statistical differences were considered significant when P < 0.05.The data are shown as the mean ± SD. Comparisons between two groups were performed using two-tailed Student P < 0.05), whereas at high doses (0.5–0.7 μg), pEASY-Blunt-M2-LEF1 decreased the promoter activity in ICP2 cells (P < 0.05) (P > 0.05) (data not shown). Collectively, these results suggest that this LEF1 binding site is involved in the regulation of MIR17HG.Our previous bioinformatics analysis showed that a potential LEF1 binding site within the MIR17HG promoter region was conserved among chicken, mouse, and human species , suggest < 0.05) . These rin vivo binding of β-catenin/LEF1 to this LEF1 binding site in ICP2 cells. ICP2 cells were transfected with pEASY-Blunt-M2-LEF1, pCMV-HA-WT-β-catenin, or pEASY-Blunt-M2/pCMV-HA. ChIP assay was performed using anti-β-catenin and anti-LEF1 antibodies as well as normal rabbit IgG control. Enriched DNA was analyzed using quantitative PCR with a specific pair of CHIP qRT-PCR primers (P < 0.01). As expected, the MIR17HG promoter fragment was not enriched in any one of the negative controls (pEASY-Blunt-M2 and pCMV-HA) (P > 0.05). Taken together, all these data suggest that LEF1 and β-catenin specifically bind to and regulate the MIR17HG promoter.To validate whether LEF1 directly binds to the potential LEF1 binding site within the MIR17HG promoter region, we performed EMSA of nuclear protein extracts from the LEF1-transfected DF1 cells with biotin-labeled wild-type (WT-LEF1) oligonucleotide probes corresponding to the MIR17HG promoter  . A speci primers . As demoP < 0.05), and combined ectopic expression of LEF1 and β-catenin further enhanced the promoter activity of pGL3-WT-MIR17HG-Luc, compared with LEF1 and β-catenin alone (P > 0.05). To validate our findings, we also performed LEF1 and β-catenin knockdown experiments. We first confirmed the knockdown efficiency of LEF1 and β-catenin at both mRNA and protein levels using qRT-PCR and Western blotting, respectively (P < 0.05)  . Taken tP < 0.01), indicating that LiCl treatment can activate Wnt/β-catenin signaling pathway. Unlike LiCl treatment, XAV-939 treatment exhibited no significant effect on Top/Fop ratio (P > 0.05) (P < 0.05), whereas LiCl treatment at a high concentration (30 mM) led to a decrease in the luciferase activity of the MIR17HG promoter (P < 0.01) (P > 0.05) (P > 0.05) , an activator of Wnt/β-catenin signaling , and XAV > 0.05) . Consist > 0.05) . As show < 0.01) . In cont > 0.05) . As expe > 0.05) . CollectP < 0.05) (P < 0.05) (P < 0.05)  and two mature miR-17-92 cluster members (miR-17-3p and miR-17-5p) in the ICP2 and DF1 cells whose Wnt/β-catenin pathway is activated or inactivated by LiCl treatment or XAV-939 treatment. Real-time RT-PCR analysis showed that 3 mM LiCl treatment increased the expression of pri-miR-17-92 and miR-17-3p by 1.36-fold and 1.30-fold compared with 3 mM NaCl-treated cells, respectively ( < 0.05) . However < 0.05) . In cont < 0.05) . Taken tP < 0.05), but not at 72 h. Conversely, 30 mM LiCl treatment significantly decreased the expression of cyclin D1 and PCNA at 24 and 72 h (P < 0.01)  . Conside < 0.01) . We concin vivo ChIP assays showed that BMP and TGF-β signaling pathways directly increases the transcription of miR-17-92 via activation of R-Smad  and high (30 mM) doses of LiCl exhibited opposite effects on cell proliferation . A previWnt signaling is one of the key signaling pathways regulating development and tightly linked to cancer. LiCl is a well-established inhibitor of Glycogen synthase kinase-3 (GSK3) and widely used to activate Wnt/β-catenin signaling in development and cancer studies . LiCl trIn conclusion, our results demonstrated that miR-17-92 cluster is a target of the Wnt/β-catenin/LEF1 pathway and that Wnt/β-catenin/LEF1 signaling pathway promotes cell proliferation at least in part via direct upregulation of miR-17-92 cluster.NW conceived and supervised the study. NW, FM, and TX designed the experiments. FM, JH, and TX performed the experiments. FM wrote the manuscript. All authors made manuscript revisions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Taken together, our study indicates that tumor cells-derived Wnt ligands stimulate M2-like polarization of TAMs via canonical Wnt/β-catenin signaling, which results in tumor growth, migration, metastasis, and immunosuppression in HCC. To block Wnts secretion from tumor cells and/or Wnt/β-catenin signal activation in TAMs may be potential strategy for HCC therapy in future.Tumor-associated macrophages (TAMs) are a major component of tumor microenvironment (TME) and play pivotal roles in the progression of hepatocellular carcinoma (HCC). Wnt signaling is evolutionarily conserved and participates in liver tumorigenesis. Several studies have shown that macrophage-derived Wnt ligands can activate Wnt signaling in tumor cells. However, whether Wnt ligands secreted by tumor cells can trigger Wnt signaling in macrophages is still elusive. In this study, we first verified that canonical Wnt/β-catenin signaling was activated during monocyte-to-macrophage differentiation and in M2-polarized macrophages. Knockdown of β-catenin in M2 macrophages exhibited stronger antitumor characteristics when cocultured with Hepa1-6 HCC cells in a series of experiments. Activation of Wnt signaling promoted M2 macrophage polarization through c-Myc. Moreover, co-culturing naïve macrophages with Hepa1-6 HCC cells in which Wnt ligands secretion was blocked by knockdown of Wntless inhibited M2 polarization in vitro. Consistently, the growth of HCC tumor orthotopically inoculated with Wntless-silenced Hepa1-6 cells was impeded, and the phenotype of M2-like TAMs was abrogated due to attenuated Wnt/β-catenin signaling in TAMs, leading to subverted immunosuppressive TME. Finally, we confirmed the correlation between M2 macrophage polarization and nuclear β-catenin accumulation in CD68 Recently, inflammation has been highlighted as the seventh hallmark of cancer, which establishes the relationship between tumor cells and the tumor microenvironment (TME)2. As a major component of TME, tumor-associated macrophages (TAMs) play a pivotal role in the progression of inflammation-related cancers, including HCC4. Many studies have indicated that TAMs promote tumor initiation, angiogenesis, metastasis, and suppression of adaptive immunity through the production of a large amount of cytokines, chemokines, growth factors and matrix metalloproteases in TME6. Indeed, infiltrated TAMs are associated with poor prognosis of HCC patients8. These studies suggest that TAMs can be a potential target for HCC therapy.Hepatocellular carcinoma (HCC) is one of the most common and aggressive inflammation-related human cancers in the world9. Under inflammatory stimulation, monocytes are recruited to injured tissue and differentiate into macrophages with differently polarized activation states. Activation with interferon-gamma (IFN-γ), or IFN-γ combined with lipopolysaccharide (LPS) polarizes macrophages into classically activated macrophages, namely M1 macrophages, which develop the proinflammatory Th1 immune response and exert tumoricidal activity by the expression of high levels of proinflammatory cytokines, such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, and high production of reactive nitrogen and oxygen intermediates (RNS and ROS), respectively. In contrast to M1 macrophage polarization, IL-4/IL-13, IL-10 or TGF-β induces macrophages to polarize into alternatively activated macrophages, referred to as M2 macrophages, which are associated with the anti-inflammatory Th2 immune response and possess protumor activity by high expression of mannose receptor (MR), arginase1 (Arg1) and Ym110. In most tumors, the characteristics of TAMs are similar to M2 macrophages in several aspects, and therefore, TAMs are also called M2-like macrophages11. Currently, the molecular mechanisms of macrophage polarization have been explained at different levels, including signaling pathways, transcription factors, and epigenetic regulation12. However, the detailed mechanisms underlying the crosstalk between tumor cells and macrophage polarization in TME remains largely unknown.TAMs possess high heterogeneity, which can be ascribed to their origin and activation status and function6. Many kinds of soluble factors, such as Wnts, are important for regulating cell−cell interaction13. Wnt ligands are secreted proteins that not only participate in cellular proliferation, migration and tissue patterning during embryonic development, but also are involved in many diseases, especially tumorigenesis16. Generally, Wnt ligands can be secreted into the extracellular milieu controlled by Wntless, and then bind to the Frizzled receptors on the signal-competent cells to induce the canonical Wnt/β-catenin signaling or noncanonical Wnt/Ca2+ signaling in paracrine/autocrine manners17. Several studies have shown an autocrine mechanism for constitutive Wnt pathway activation in human cancer cells including breast cancer, ovarian cancer, and non-small cell lung carcinoma19. In contrast, Binder’s and Pollard’s groups found that a paracrine Wnt signaling loop exists between breast tumor cells and TAMs using in vitro and in vivo assays, as Wnt ligands can also be expressed by macrophages21. Moreover, Cosin-Roger et al. reported that Wnt ligands from M2 macrophages activate Wnt signaling in intestinal epithelial cells22. It is known that Wnt/β-catenin signaling plays important roles in liver development, regeneration, and cancer, and that Wnt ligands and receptors can be expressed by various hepatic cell types, such as hepatocytes and Kupffer cells (KCs) that are one kind of tissue resident macrophages23. Recently, Boulter et al. have found that macrophage-derived Wnt3a opposes Notch signaling to promote hepatic progenitor cell specification to hepatocytes in chronic liver diseases24. Therefore, we wondered whether hepatic tumor cells-derived Wnt ligands can trigger Wnt/β-catenin signaling in macrophages through a paracrine manner.Growing evidence shows that crosstalk between tumor cells and macrophages is involved in tumor progressionIn this study, we report that the Wnt/β-catenin signaling was activated during monocyte-to-macrophage differentiation and in M2 macrophages. Activated Wnt/β-catenin signaling promoted M2 macrophage polarization, which elicited M2-like phenotype of TAMs in coculture with Hepa1-6 HCC cells. Further investigation showed that Wnt/β-catenin signaling regulated M2 macrophage polarization through c-Myc. Blocking Wnt ligands secretion from Hepa1-6 cells by Wntless knockdown inhibits M2 macrophage polarization in vitro and reduces tumor growth in vivo by reprogramming the tumor immune microenvironment. Finally, the correlation between M2 macrophage polarization and nuclear β-catenin accumulation in TAMs was verified in HCC patient biopsies. Taken together, our study for the first time showed that hepatic tumor cells promoted M2 macrophage polarization through Wnt/β-catenin signaling in a paracrine manner. Blocking Wnt secretion from hepatic tumor cells and/or Wnt/β-catenin signal activation in TAMs may be a promising strategy for liver cancer therapy.27. Therefore, we assessed the possibility whether Wnt/β-catenin signaling is involved in the differentiation of monocytes into macrophages in mice. CD11b+ monocytes were sorted from mouse bone marrow (BM) by magnetic activated cell sorting, and then were induced into macrophages with M-CSF stimulation for 7 days. The purity of the monocytes and macrophages was approximately 98% by FACS assay  were polarized to M1 or M2 phenotype under LPS + INF-γ or IL-4 stimulation, respectively, we examined the mRNA expression of Wnt receptors and downstream genes of Wnt signaling in M0, M1 and M2 BMDMs by qRT-PCR. We found that the expression level of Wnt receptors such as Fzd7 and Fzd9 was lower in M1 macrophages but comparable in M2 macrophages, suggesting that Wnt signaling could be involved in macrophage polarization Fig. . Indeed,28, or lithium chloride (LiCl)29 which is a GSK-3β inhibitor, were added into BMDM culture followed by LPS + INF-γ treatment for 24 h. The result showed that the expression of M1 macrophage surface markers such as TNF-α and IL-12 was significantly reduced, and the functional marker iNOS was also reduced slightly under M1 status with Wnt3a or LiCl treatment - or siR-transfected differentially polarized macrophages by a mixed lymphocyte reaction assay. The FACS assay showed that si-β-catenin(S1)-transfected M0 or M2 macrophages promoted CD8+ T-cell proliferation more strongly than siR-transfected M0 or M2 macrophages  efficiently repressed β-catenin expression in BMDMs by qRT-PCR and western blotting Fig. . Becauseges Fig. . Taken t23, we postulated that activated Wnt/β-catenin signaling in TAMs might be initiated by Wnt ligands secreted from Hepa1-6 cells in TME. To confirm this possibility, we first examined the mRNA levels of Wnt ligands in differentially polarized macrophage, which showed that the expression of several Wnt ligands, such as Wnt3, 3a, 4, 10b, and 11, were relatively higher in M2 macrophages than that in M1 macrophages, and the expression of Wnt2, 6, 11, and 16 were relatively higher in M1 macrophages compared with that in M0 macrophages  is an evolutionarily conserved multipass transmembrane protein that is specially required for Wnt ligands secretion+ TAMs in sh-WLS HCC-bearing mice decreased by Ki67 immunofluoresence staining of tumor sections  increased while that of CD25+Foxp3+ Treg cells decreased , β-catenin and M2-related markers MR or Arg1 in 25 frozen tumor sections by immunofluorescence staining. As shown in Fig. +Arg1+ or CD68+MR+ M2-like TAMs infiltrated into HCC tumors and some of them exhibited an obvious nuclear localization of β-catenin. Data quantification showed that the level of nuclear β-catenin was positively correlated with the Arg1 or MR expression in CD68+ macrophages in HCC patient biopsies  in 30 μl Matrigel  were orthotopically inoculated into the left liver lobes of mice under anesthesia with 0.6% pentobarbital sodium solution . After 3 weeks, mice were sacrificed. Tumor weight was measured. And then tumor was minced for further analysis. All mouse experiments were conducted according to a guideline from the Animal Experiment Administration Committee of the Fourth Military Medical University and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health .C57BL/6 mice (8–10 weeks of age) were maintained in a specific pathogen-free (SPF) environment. The liver tumor model was established using C57BL/6 mice as follows: for the orthotropic HCC model, Hepa1-6 cells  containing 10% fetal calf serum  and 25 ng/mL murine macrophage-colony stimulating factor  for 7 days to obtain BMDMs. M1 or M2-polarized macrophages were induced by adding LPS  + rIFN-γ  or IL-4  into the cultured BMDMs for 24 h, respectively. In some experiments, after being treated with Wnt activator , BMDMs were stimulated with LPS + rIFN-γ or IL-4 for further analysis. For transfection, cultured BMDMs were transfected with 100 nM β-catenin or c-Myc small interfering RNA (siRNA-β-catenin or siRNA-c-Myc) or randomized siRNA control (siR)  using the Lipofectamine LTX  according to the protocol. Total RNA or proteins were extracted 48 h after the transfection. All siRNA sequences are shown in Supplement Table BMDMs were induced and cultured as previously describedl-glutamine (Gibco) and 1% penicillin/streptomycin (Gibco) at 37 °C in a 95% air-5% CO2 incubator. In some cases, the CM from cultured Hepa1-6 cells was collected and added into BMDMs to induce M2-like macrophages for 24 h. Meanwhile, the inhibitor of Wnt signaling, ICG001 , was supplied into the medium. Then, cells were collected for further experiments.The murine hepatoma cell line (Hepa1-6) was originally purchased from ATCC . Cells were cultured in DMEM supplemented with 10% FCS, 2 mM Hepa1-6 cells were seeded into six-well plate at a density of 200 cells per well and cultured with CM, which was collected from cultured M2 macrophages transfected with si-β-catenin or randomized siRNA control (siR) for 2 weeks. After that, Hepa1-6 cells were washed with PBS twice, fixed with 4% paraformaldehyde (Sigma) and stained with 1% crystal violet . The number of colonies was counted.6/well and grown to 80–90% confluence. The cells were scratched with a sterile 200 µL micropipette tip and the debris were washed away. Then the cells were cultured with CM as described above for 0, 8, and 16 h. The cell migration distance between the two scratches was recorded and measured with ImageJ software. The cell migration distance was calculated as follows: average gap area at 0 h – average gap area at 8/16 h.Hepa1-6 cells were seeded into 12-well plates at a density of 2 × 104 Hepa1-6 cells were suspended in 200 µL serum-free DMEM and plated in upper chamber without Matrigel (for the migration experiment) or with Matrigel (for the invasion experiment) coated (Corning). After incubating at 37 °C for 24 h (for the migration experiment) or 48 h (for the invasion experiment), the non-invading cells were scrubbed away by a cotton swab. The cells that had penetrated through the filter were stained with crystal violet and counted under a microscope. Ten randomly selected fields were used to count the number of migrating cell number in each insert.BMDMs were seeded into the 12-well plate and transfected with si-β-catenin or siR as described above. And then BMDMs were stimulated with IL-4 for 24 h. Meanwhile, 4 × 1048. BMDMs were seeded into 24-well plates. After transfection with si-β-catenin or siR, macrophages were stimulated with PBS (M0), LPS + INF-γ (M1) or IL-4 (M2) for 24 h. Then, cells were irradiated with 20 Gy X-rays. Naïve T cells were negatively sorted from the lymph nodes of allogeneic mice using biotin-labeled B cell- and myeloid cell-specific antibodies, followed by magnetic Streptavidin Particles Plus . T cells were labeled with carboxyfluorescein diacetatesuccinimidyl ester  and added into irradiated differentially polarized BMDMs at a ratio of 5:1.5 days later, nonadherent cells were harvested and stained with APC anti-CD8 antibody (BD Pharmingen). Proliferation of CD8+ T cells was analyzed by FACS.A mixed lymphocyte reaction was performed as previously describedTM flow cytometer (BD Immunocytometry Systems) were applied for sample analysis. FACS data were analyzed using Flowjo vX.0.6 software .Tumors tissues were dissected from PBS-perfused mice. After measuring their weight, tumors were finely chopped and incubated with 1 mg/mL collagenase type V (Sigma) and 100 µg/mL DNase I  for 1 h at 37 °C. Digested tissues were mashed through cell strainers (70 µm) and subjected to removal of red blood cells. The harvested single cells were incubated with relevant antibody cocktails for 20 min on ice in the dark following the standard protocols. Biotin-labeled primary antibodies were detected with PE-streptavidin as the secondary antibody. Cell viability was evaluated using 7-AAD (BD Biosciences). For intracellular staining, cells were permeabilized with Permeabilization Buffer (Invitrogen) and stained with PE-conjugated primary antibodies. FACSCaliburThe primary Kuppfer cells (KC) from the liver of wild-type mice or TAMs from orthotopic HCC-bearing mice were sorted using a FACSAriaII flow cytometer (BD Biosciences). The sorted cells were subjected to preparation of total mRNA for subsequent analysis of gene expression. The information of all the antibodies used is shown in Supplement Table Total RNA of cells was extracted using TRIzol (Invitrogen) or LS TRIzol reagent (Invitrogen) according to the manufacturer’s instruction. Reverse transcription was performed using a kit with random primers , and then real-time PCR was carried out using QuantiTect SYBR Green PCR Kit (Takara) on the ABI PRISM 7500 real-time PCR system (Applied Biosystems), with β-actin as an internal control. All the primers used are shown in Supplement Table For cell immunofluorescence, cultured cells were grown on cover slides until confluence, and fixed in 4% paraformaldehyde for 15 min, followed by three washes with PBS. Cells were incubated with primary antibodies including anti-F4/80, anti-β-catenin, anti-MR, anti-Arg1 or Ki67 followed by secondary antibody staining. Nuclei were counterstained with Hoechst 33258 (Sigma). Photographs were taken using a laser scanning confocal microscope .Liver sections from biopsies of HCC patients were prepared according to standard procedures. The primary antibodies included anti-CD68, anti-β-catenin, anti-MR, or anti-Arg1 followed by secondary antibodies staining. All antibodies used are listed in Supplement Table Western blotting was performed according to standard procedure. Each 40 µg protein samples were loaded and separated by SDS-PAGE, and subsequently transferred onto polyvinylidene fluoride membranes. The protein-blotted membranes were incubated with primary antibodies overnight at 4 °C. HRP-conjugated secondary antibodies were incubated at room temperature for 1 h. The chemoluminescence signals were developed with Pierce ECL Western Blotting Substrate  and detected using the ChemiScope imaging system . The protein expression level was quantified and normalized to β-actin as an internal reference with ImageJ software (NIH). The information of all antibodies used is listed in Supplement Table The shRNA targeting the Wntless (sh-Wntless) and the control shRNA were inserted into the lentiviral expression vector . In addition, viral particles were prepared by transfecting HEK293T. Hepa1-6 cells were transduced following the recommended protocols. The shRNA sequence against Wntless was shown in Supplement Table t test, paired t test or one-way ANOVA with Tukey’s multiple comparison test was used for the statistical analysis. P < 0.05 was considered statistically significant.All the images were quantified by Image Pro Plus 5.1 software (Media Cybernetics Inc.). Data were analyzed using Graph Pad Prism 5 software (San Diego). An unpaired Student’s Supplement Material


We also successfully labeled common input of the lateral geniculate nucleus to both cortico-cortical layer 5 neurons and inhibitory neurons of the mouse V1 with multiplex RVΔG tracing. These oEnvA/oTVA, oEnvB/oTVB, and oEnvE/oTVE systems allow for differential labeling of distinct circuits to uncover the mechanisms underlying parallel processing through independent circuits and integrated processing through interaction between circuits in the brain.Neural circuits interconnect to organize large-scale networks that generate perception, cognition, memory, and behavior. Information in the nervous system is processed both through parallel, independent circuits and through intermixing circuits. Analyzing the interaction between circuits is particularly indispensable for elucidating how the brain functions. Monosynaptic circuit tracing with glycoprotein (G) gene-deleted rabies viral vectors (RVΔG) comprises a powerful approach for studying the structure and function of neural circuits. Pseudotyping of RVΔG with the foreign envelope EnvA permits expression of transgenes such as fluorescent proteins, genetically-encoded sensors, or optogenetic tools in cells expressing TVA, a cognate receptor for EnvA. Trans-complementation with rabies virus glycoproteins (RV-G) enables trans-synaptic labeling of input neurons directly connected to the starter neurons expressing both TVA and RV-G. However, it remains challenging to simultaneously map neuronal connections from multiple cell populations and their interactions between intermixing circuits solely with the EnvA/TVA-mediated RV tracing system in a single animal. To overcome this limitation, here, we multiplexed RVΔG circuit tracing by optimizing distinct viral envelopes (oEnvX) and their corresponding receptors (oTVX). Based on the EnvB/TVB and EnvE/DR46-TVB systems derived from the avian sarcoma leukosis virus (ASLV), we developed optimized TVB receptors with lower or higher affinity (oTVB-L or oTVB-H) and the chimeric envelope oEnvB, as well as an optimized TVE receptor with higher affinity (oTVE-H) and its chimeric envelope oEnvE. We demonstrated independence of RVΔG infection between the oEnvA/oTVA, oEnvB/oTVB, and oEnvE/oTVE systems and  The function of the nervous system arises from complex interactions between networks of neurons composed of multiple cell types. Each cell type has its own molecular, morphological, neurophysiological, and anatomical properties and organizes unique neural circuits. The wiring patterns of individual cell types underlie how neural circuits process and represent information. Detailed information on the cell types and their connectivity, in addition to the spatiotemporal patterns of activity in neural circuits, is essential for understanding how the brain functions. Parallel information processing through independent circuits is a commonly-used strategy in the brain  have been used to reveal neuronal connectivity in the mammalian nervous system . In conjunction with different colors of reporter fluorescence such as GFP, RFP, BFP, and iRFP, and functional fluorescent probes including genetically encoded sensors for CaE.coli  was transformed with AAV plasmids, and chemically-competent XL10-Gold E. coli  was used for the other plasmids. The E. coli was grown in LB medium  on an LB plate containing ampicillin (100 μg/ml). Plasmids were inspected by diagnostic digestions with appropriate restriction enzymes and sequenced before virus production.All plasmid construction using polymerase chain reaction (PCR) was performed with PrimeSTAR Max DNA Polymerase  on a PCR thermal cycler . Plasmids and PCR products were digested with appropriate restriction enzymes  at optimal conditions. Plasmid and PCR fragments were assembled with either NEBuilder HiFi DNA Assembly Master Mix  or DNA Ligation Kit Mighty Mix (TaKaRa). Chemically-competent Stbl3 2 and 95% air at 37°C.HEK293t cells were obtained from RIKEN Cell Bank . HEK-TVA, HEK-TVB, BHK-T7, BHK-EnvA, BHK-EnvB, and B7GG cells were gifted by Dr. Callaway  or 1.0 × 1011–1.8 × 1014 viral genome/ml quantitative PCR . Tlx3-Cre mice (PL56) were obtained from the Jackson Laboratory . Genotyping for Tlx3-Cre mice was performed according to the protocol of Mutant Mouse Resource and Research Center: UC Davis. These animals were maintained in a temperature-controlled room (24 ± 1°C) under a 12 h light/dark cycle withvia a small incision, and a small hole was drilled for injection. The virus was loaded into glass pipettes , pulled on a micropipette puller  and grinded on a microgrinder . Before injection of the virus, the pipette was retained in the brain for 5 min after penetration. Three hundered to five hundered nanoliter of virus was injected into the following locations at a rate of 100 nl/min: the V1 , V2L , and SC  using a picospritzer . After completion of the injection, the pipette was retained in the brain for another 5 min. No mouse that received viral injection in the brain showed abnormal behavior.Viral injections were conducted according to the procedures described previously  and transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). The brains were post-fixed with 4% paraformaldehyde in PBS and cryopreserved overnight in 2% paraformaldehyde/15% sucrose in PBS and then maintained in 30% sucrose in PBS. The brains were sectioned on a freezing microtome  at 50 μm thickness. For enhancement of native signals, some brain sections were processed for immunostaining as described previously , oEnvB-RVΔG-DsRed (red), and oEnvE-RVΔG-GFP  and indicators is not useful, as many fluorescent indicators such as GCaMP, XCaMPs, jRGECO, iGluRSnFr, and dLight1 are of the same color  as a reporter, the brightest infrared fluorescent protein at present . These results indicate that oEnvA-RVΔG-GFP specifically infected the oTVA-L-mCherry-expressing neurons in layer 5 of the mouse cortex.We next evaluated the + cells expressed GFP in layer 5. All GFP+ cells were positive for iRFP670 in layer 5 . These results indicate that oEnvA-RVΔG-GFP specifically infected the oTVA-L-iRFP-expressing neurons in vivo. No infection with oEnvA-RVΔG-DsRed was observed in the absence of oTVA-L in mice . As TVA950 was distributed on the membrane (950 to generate a TVBS3-TVA chimera receptor (S3-TVA chimera receptor was fused to tagBFP and expressed in the cell membrane without aggregating inside the cell (S3-TVA-tagBFP and then we applied oEnvB-RVΔG-DsRed to the transfected cells (S3-TVA-tagBFP-expressing cells were positive for DsRed. TVBS3-TVA-tagBFP had higher infection efficiency by oEnvB-RVΔG compared to TVBS3-tagBFP. We termed the TVBS3-TVA receptor “oTVB-H” (optimized TVB with the highest affinity). We further assessed the cross-infectivity of oTVB-H to oEnvX-pseudotyped RVΔG. Each oEnvX-pseudotyped RVΔG was applied to oTVB-H-expressing HEK293t cells  to the V1 of Tlx3-Cre mice . oEnvB-Rnegative . Tlx3-Cr950 and TVA950E66T  that can recognize and bind to ASLV-B  at CRD2, and TVBr2 has two mutations at CRD2 (S62C) and CRD3 .To overcome this issue, we attempted to clearly visualize the starter cells with fluorescent reporters by developing a low-affinity type TVB receptor, as shown in the cases of TVAA-tagBFP . CompareBS3ΔCRD3 . To evalse cells  because TVA-iRFP . TVBS3ΔCin vivo viral targeting to specific cell types and unambiguous visualization of the infected cells. We injected Cre-dependent AAV expressing oTVB-L-tagBFP (AAV2/9-CAG-DIO-oTVB-L-tagBFP) into the V1 of Tlx3-Cre mice to restrict oTVB-L-tagBFP expression to layer 5 neurons  in the V1 of layer 5-specific Tlx3-Cre mice, as previously performed with oTVA-L and oTVB, and then injected oEnvE-RVΔG-GFP into the same location  and single mutated oTVE-H receptors  fused to tagBFP . GFP-positive and H2B-tagBFP-negative neurons were observed outside layer 5 of the cortex. This is because leak expression of oTVE-H derived from AAV-DIO vectors induced oEnvE-RVΔG-GFP infection. These results suggest that the 2A-system permits labeling and detection of the starter neurons with a fluorescence reporter and that only a small amount of oTVE-H is needed for the infection of oEnvE-RVΔG even at a barely visible expression level of its reporter fluorescence.Clear labeling of starter neurons is essential to determine the post-synaptic neurons in rabies tracing experiments, although the cells expressing oTVE-H-mCherry can be infected with oEnvE-RVΔG even when its expression is too low to be detected. It is conceivable that oTVE-H is inefficiently expressed in the mouse brain and its fused-reporter signal is barely detected. Thus, to reliably visualize target cells expressing oTVE-H, we first used a 2A-mediated bicistronic expression system . Compared to the 2A system that enables the bicistronic expression of two genes at the same expression level  system, which can adjust oTVE-H expression to a lower level . We gene+ presynaptic cells in the V1 and dorsal lateral geniculate nucleus . No co-infection was observed between the oTVA-L-iRFP- and oTVB-L-tagBFP-expressing AAV  were injected into the lateral higher visual area (V2L: the putative area LM) to introduce both TVA and oG to V2L-projecting layer 5 neurons  in layer 5 of the V1. We also found GFP+ and DsRed+ cells, indicating that common input was detected as co-infection with both oEnvA-RVΔG-DsRed and oEnvB-RVΔG-GFP. Interestingly, GFP+/DsRed+ neurons were distributed in the V1  fused to TVA950 were invisible. For example, in transgenic mice expressing Cre in a specific cell population, Cre-negative cells can express a small amount of TVA950 due to leak expression, which leads to non-specific infection with EnvA-RVΔG. However, when using TVA950E66T-mCherry for rabies viral targeting in Cre-expressing transgenic mice, oEnvA-RVΔG cannot infect the cells expressing a small amount of TVA950E66T-mCherry, which are invisible under a fluorescent microscope because of the low affinity of TVA950E66T to oEnvA  is ideal for rabies viral tracing.TVA fused to a fluorescent protein has been used to visualize TVA-expressing starter cells: TVA800, TVBS3, and DR46-TVB  howOptical techniques have become indispensable for neuroscientific research in recent decades (Yang and Yuste, To overcome these limitations, chemical tags could be a viable alternative to fluorescent proteins. Genetically-encoded chemical tags, such as Halo-tag, SNAP-tag, CLIP-tag, and TMP-tag, were labeled with synthetic fluorophores (Gautier et al., Rabies viral vectors are powerful tools for labeling synaptically-connected neurons throughout the brain. Despite the strength in anatomical studies, application of RVΔG tracing in physiological and behavioral studies remains limited because of its toxicity (Osakada et al., Revealing parallel processing through independent circuits and integrated processing through interaction between circuits is pivotal in understanding how the brain functions. The multiplex RV tracing that we developed in the present study will facilitate the in-depth understanding of the principles of neural circuits and computations in the brain.All datasets generated for this study are included in the article/The animal study was reviewed and approved by the Animal Care and Use Committee in Nagoya University.TS performed experiments. TS, NM, and FO analyzed the data. TS, NM, AA, and FO wrote the manuscript. FO designed the study and supervised this study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Endometriosis is an oestrogen‐dependent disease, and epithelial‐mesenchymal transition (EMT) is involved in the process of endometriosis. Whether oestrogen could induce EMT in endometriosis remains largely unknown. Here, we reported that up‐regulated expression of EMT markers in ovarian chocolate cyst is accompanied by high expression 17β‐hydroxysteroid dehydrogenase 1 (17β‐HSD1), and exposure of primary human endometrial epithelial cells to oestradiol conditions could promote EMT occurrence and activate both β‐catenin and Snail signalling. Furthermore, we found nuclear β‐catenin and Snail expression was closely linked in ovarian endometriosis, and β‐catenin knockdown abrogated oestrogen‐induced Snail mediated EMT in vitro. This is due to that β‐catenin/ TCF‐3 could bind to Snail promoter and activate its transcription. These results suggested that β‐catenin signalling functions as the Snail activator and plays a critical role in oestradiol‐induced EMT in endometriosis. And β‐catenin signalling activating Snail promoter functions a crucial role in oestrogen‐facilitated migration and invasion in endometrial epithelial cells.22.1Patients, from Department of Obstetrics and Gynecology, Union Hospital between June 2013 and January 2014, signed informed consent approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (IORG No: IORG0003571). In this study, samples of normal endometrium (controls), paired eutopic endometria and ovarian chocolate cyst (endometriotic tissue samples) were obtained from 21 healthy women and 21 women with ovarian endometriosis, respectively. Primary endometrial epithelial cells were obtained from another 32 healthy women. Endometriosis was suspected from the results of either physical examination or ultrasonography. Endometriosis patients were confirmed by laparoscopic surgery. The staging of endometriosis is based on the revised American Society for Reproductive Medicine (rASRM). All of the patients did not receive any hormonal treatment for at least 6 months before specimen collection. All samples were histologically confirmed by the pathologist. In Table 2.2The procedures of IHC were carried out as previously described.2.3The protocol of primary human endometrial epithelial cells (EECs) culture was confirmed as previously described.2 dissolved in dimethyl sulfoxide (DMSO) was purchased from Sigma‐Aldrich (E‐2758). ICI 182.780 (ICI), an oestrogen receptor antagonist, dissolved in DMSO, was purchased from CAYMAN (CAS 129453‐61‐8).E2.4Ct value was used to calculate the expression levels of target genes.RNA extractions and real‐time quantitative PCR (RT‐PCR) analyses were confirmed as previously described.2.5The extractions of protein and Western blot protocol were confirmed as previously described.2.65 Cells/mL were collected and resuspended in 200 µL serum‐free DMEM. Then, cells were seeded on the each polycarbonate filters above the 24‐well plate which contains 500 µL 20% FBS‐DMEM. After incubating at 37°C for 24 hours, the cells were fixed and stained for 15 minutes in a 0.1% crystal violet solution in PBS. The number of cells on the underside of each insert was determined using light microscopy . Five randomly selected fields were counted per insert.Polycarbonate filters with 8 μm pore size membranes (Corning Costar) combined with 24‐well culture plates were used for migration (plain) and invasion (matrigel‐coated) assay. 102.72 at 37°C.Transfection was used by Lipofectamine 2000 (Invitrogen Life Technologies). A certain proportion of siRNA and Lipofectamine 2000 were mixed in accordance with the instruction manual. Ishikawa cells were transfected with the mixture, and cell culture medium was changed to DMEM/F‐12 supplemented with 5% FBS after 6 hours. Then, the transfected cells were cultivated in 5% CO2.85 Ishikawa cells were cultured in 24‐well plates and cotransfected with pGL3B and Renilla plasmid. Luciferase activity was calculated by the Dual‐Luciferase Reporter Assay System (Promega) after 48 hours of transfection.The human Snail promoter (~2.1 kb) spanning from −2084 to +50 from the transcriptional initiation to the pGL3‐Basic (pGL3B) luciferase vector was cloned. We used the construct together with the β‐catenin or TCF3 expression vector (Genechem) together with the Snail promoter to cotransfect Ishikawa. Cotransfections were carried out in 24‐well plates. 1 × 102.9−6 mol/L E2 (Sigma‐Aldrich) or the control DMSO for 48 hours. EECs were fixed by 4% cold paraformaldehyde for 20 minutes when grown on a cover sheet and fused to 95%‐100%. Then, EECs were permeable for 10 minutes by 0.2% Triton X‐100 and washed by PBS three times. EECs were blocked with 5% bovine serum albumin (BSA) for 30 minutes at RT and incubated with primary rabbit antibodies against β‐catenin  and mouse antibodies against Snail  overnight at 4°C. Finally, the EECs were incubated with appropriate secondary antibodies conjugated to fluorescein isothiocyanate (FITC) or Texas Red (KPL) at RT in a humidified chamber. The specimen was stained with nuclei by added DAPI (Sigma‐Aldrich) for 10 minutes. Then, the acquired image was observed under a fluorescence microscope (Olympus).EECs were cultured on cover sheet placed inside 6‐well plates and stimulated with 102.102 or DMSO for 48 hours. ChIP was carried out on the basis of the product manual for the ChIP kit (Millipore). Antibody against β‐catenin  was used. PCR was then performed by using a set of primers designed to amplify the Snail promoter TCF binding sites (TBS). The primer sequences are listed: 5′‐ACTATGCCCACCGACCCT‐3′ and 5′‐CCAGACCTTTCCCACCTT‐3.Before the chromatin immunoprecipitation assay (ChIP), cells were dealt with E2.11t test or one‐way analysis of variance was applied for the comparison of different groups. Spearman's rank correlation coefficient was applied for rank correlation. Statistical significance was set at P‐value <.05. All experiments were performed in triplicate.All values were shown as the mean ± standard deviation (SD) or mean ± standard error of the mean (SEM). Fisher's exact tests were applied for the comparison of dichotomous variables. Unpaired 33.1IHC analysis of EMT markers in eutopic endometrium (EU) and ovarian chocolate cyst (OC) was performed in tissues from 21 patients with endometriosis and 21 normal control (NC) cases. Representative IHC results are shown in Figure Positive E‐cadherin expression was present in cell membranes of the epithelial glands in majority of the NC samples (85.7%). The trend of positive E‐cadherin staining in the EU samples (90.5%) was not statistically different from that of the NC samples. However, the endometrial epithelial cells of OC samples showed negative E‐cadherin staining (52.4%). For vimentin, proportion of vimentin‐positive epithelial cells was significantly increased in the OC compared with the NC cases (66.7% vs 14.3%), and the difference between the NC group and EU group was not significant.2).β‐catenin and Snail expression was also detected in the nuclei of epithelial and stromal cells. They were significantly increased in OC group compared with EU group  and NC group  . Immunostaining scores for E‐cadherin in EECs were apparently decreased in the 10−8 mol/L E2 group (3.333 ± 2.082) compared with the control (8.667 ± 2.082), and most obviously in the 10−6mol/L E2 group (2.667 ± 1.528). During EMT process, epithelial cells obtain the ability of distant migration and metastasis and show morphological changes.2‐treated human endometrial epithelial cells were more migratory and invasive compared with control cells . Also, E2 (10−6mol/L) induced EECs to shift from a cobblestone‐like appearance  to an elongated spindle‐shaped cell . And cytokeratin expression of the EECs was apparently decreased in the E2 treated group .Endometriosis is known as oestrogen‐dependent disease.−8 mol/L and 10−6mol/L E2 groups , in accordance with the immunostaining result  in the 10−6mol/L E2 group . These results demonstrated that E2 induces EMT in a dose‐dependent manner in EECs, and the effect was maximal at 10−6mol/L E2 at 48 hours. These findings suggested that oestradiol, a dominant factor for endometriosis, could cause EMT in human epithelial cells by up‐regulating the expression of β‐catenin and Snail.Furthermore, down‐regulated E‐cadherin and up‐regulated vimentin was detected in the 103.32 without or with ICI . RT‐PCR revealed that decreased E‐cadherin mRNA expression in E2 group was reversed by ICI  mRNA was indeed antagonized by ICI. Furthermore, we investigated the effect of E2 on β‐catenin signalling activity. As shown in Figure 2 stimulation and apparently also abolished by the ICI. Above all, these results suggested oestradiol may up‐regulate EMT‐related genes and stimulate β‐catenin signalling activation with the involvement of ERs.A classic genomic mechanism of oestradiol is to stimulate oestrogen receptors (ERs) to induce their target genes expression.I Figure A. Meanwhl Figure B‐D mRNA 3.4R = 0.2521, P = NS) fractions. There was a positive correlation between the nuclear β‐catenin and Snail expression levels in the EU group  and the OC group  respectively (data were not shown). Altogether, these results suggest that the positive relationship between β‐catenin and Snail signalling pathways in the ovarian endometriosis.β‐Catenin and Snail could both regulate the expression of the above EMT markers, and we want to make clear their relationship.3.52‐induced EMT, Ishikawa cells were transfected with β‐catenin siRNA and Snail siRNA for 24 hours and were incubated with E2 for another 24 hours. As shown in Figure 2‐induced overexpression of Snail was abrogated by β‐catenin siRNA, and β‐catenin protein expression was not changed by adding Snail siRNA, which demonstrated that the expression of Snail is relevant to the expression of β‐catenin. Furthermore, β‐catenin siRNA decreased the E2‐induced overexpression of vimentin and lower expression of E‐cadherin according to the Western blotting results. Meanwhile, the phenomenon was more obvious when the cells were transfected with β‐catenin siRNA and Snail siRNA simultaneously. In Figure 2‐induced Snail expression and EMT occurrence.Ishikawa cells are a well‐differentiated endometrial adenocarcinoma cell line that retains the endometrial epithelial phenotype and displays apical adhesiveness and expression profiles of different molecules similar to those of the endometrium under the control of oestradiol and progesterone.3.62 for 48 hours. We hypothesized that β‐catenin might up‐regulate Snail activity through binding to its promoter and activating its transcription. We cloned the human Snail promoter (~2.1 kb) spanning −2084 to +50 from the transcriptional start site into the VCG pGL3‐Basic (pGL3B) luciferase vector. As shown in Figure β‐Catenin functions as a downstream effector of Wnt signalling, playing a key role cell growth and development. Wnt signalling activation could lead to β‐catenin dephosphorylation and accumulation in the nucleus., the transcriptional activation of the Snail promoter (−2084/+50) was markedly increased by β‐catenin/TCF3 overexpression compared with Snail promoter (−210/+50). To further elucidate which binding site was the most critical for Snail induction, the binding sites at positions −1942 bp~‐1927 bp were mutated. We found that the increased luciferase activity of oestrogen and β‐catenin expression vectors on the Snail reporter was attenuated by the mutation of the binding site at positions −1942 bp ~ −1927 bp  family. Finally, the compound activates the transcription of their target genes. TCF3 is one of the most important members of TCF/LEF family.r −2084/+0 was marr −2084/+0 was mar42 treatment. Secondly, nuclear β‐catenin and Snail were coexpressed in ovarian endometriotic tissue, and β‐catenin regulated E2‐induced Snail expression in vitro. Thirdly, β‐catenin could bind to the promoter of Snail and mediate its transcription, which stimulated by E2.In this passage, we propose that the β‐catenin/Snail interaction plays an important role in oestrogen‐induced EMT in EECs and contributes to the development of ovarian endometriosis. Firstly, down‐regulated E‐cadherin expression and up‐regulated vimentin expression were observed in the epithelial cells of ovarian endometriotic tissues, and the alternations of these EMT markers were related with EWang et al reported that oestrogen could induce EMT and promote tumour growth in breast cancer.In order to explore the mechanism how oestradiol promote EMT in ovarian endometriosis, we extracted EECs from human endometrium and assessed our conjecture. Oestradiol could also apparently contribute to these changes in isolated primary epithelial cells in vitro. And this effect was reversed by ICI, further indicating oestrogen receptors involved in EMT process. ICI is the antagonist for oestrogen receptors, ERα and ERβ, and which one is responsible for the process needs more research.Accumulating evidence shows that Wnt/β‐catenin signalling plays a critical role in EMT regulation by down‐regulating the expression of E‐cadherin, which subsequently leads to β‐catenin release and activation.In this study, we first confirmed a positive correlation between the expression levels of β‐catenin and Snail in eutopic and ectopic endometriotic tissue from ovarian endometriosis patients. Moreover, β‐catenin knockdown could abolish the effects of oestradiol on the expression of Snail, E‐cadherin, vimentin and invasiveness ability of endometrial epithelial cells. Oestradiol could promote the increased nuclear expression of Snail through inducing nuclear translocation and activation of β‐catenin in EECs. Hence, the interaction of β‐catenin with Snail makes an important impact in oestrogen‐induced EMT during the development of endometriosis.2 could activate β‐catenin signalling and increase the invasiveness of endometrial stromal cells (HECSs).2 could also increase dephosphorylated β‐catenin expression and nuclear translocation in EECs. In addition, the expression of Snail stimulated by E2 depended on dephosphorylation of β‐catenin/TCF‐3 which bound to the Snail promoter and up‐regulated its transcription, as evidenced by promoter activity assays and chromatin immunoprecipitation assays. These data prove a molecular mechanism to interpret a functional link between β‐catenin and Snail in ovarian endometriosis.β‐Catenin, as an important molecule in canonical Wnt signalling, interacts with T‐cell factor (TCF) or lymphoid enhancer factor (LEF) transcription factors in nucleus to induce cellular adhesion, tissue morphogenesis and development of tumour.It is the first to illustrate β‐catenin/ Snail signalling in the development of ovarian endometriosis. We get a conclusion that oestradiol promotes β‐catenin dephosphorylation and activation, which subsequently leads to the up‐regulation of Snail transcription and expression. Overexpressed Snail interacts with the proximal E‐boxes of the E‐cadherin promoter, causing decreased expression of E‐cadherin, which could further promote β‐catenin release and activation in a positive feedback manner.2‐induced EMT in ovarian endometriosis is the major purpose of our research. These results reveal new highlights on pathophysiology of endometriosis and provided direction to investigate potential therapeutic strategies.In conclusion, highlighting the crucial role of β‐catenin/Snail in ENo potential conflict of interest was reported by the authors.Yi Liu and Zhibing Zhang conceived and designed the experiments. WenqianXiong, Ling Zhang executed the experiments. WenqianXiong and Ling Zhang analysed the data. WenqianXiong wrote the article, and all the other authors are contributed to the manuscript. Haitang He, Na Li and Yu Du are responsible for collecting specimens. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


We investigated cell viability, the transcription and translation levels of Wnt/β-catenin signaling-related genes, the extent of methylation at CpG islands of the FZD1 promoter, and the osteogenic and adipogenic differentiation abilities of MSCs from the control group and from the ONFH group treated with or without 5′-Aza-dC. According to the results, MSCs from the ONFH group showed a reduced proliferation ability, low transcription and translation levels of FZD1, inhibition of the Wnt/β-catenin signaling pathway, weakened osteogenesis and enhanced adipogenesis ability. Aberrant CpG island hypermethylation of FZD1 was observed in the ONFH group. Treatment with 5’-Aza-dC resulted in de novo FZD1 expression, reactivation of the Wnt/β-catenin signaling pathway and promotion of osteogenesis. Taken together, our study not only provides novel insights into the regulation of the Wnt/β-catenin signaling pathway in this disease but also reveals potential for the use of demethylating agents for the treatment of GC-associated ONFH.The Wnt/β-catenin signaling pathway is associated with the pathogenesis of steroid-induced osteonecrosis. Our investigation studied whether aberrant CpG island hypermethylation of the FZD1 gene was present in patients with osteonecrosis of the femoral head (ONFH), which results in Wnt/β-catenin signaling inactivation and subsequent cell dysfunction. Bone marrow was collected from the proximal femurs of patients with steroid-associated ONFH ( 3) to the DNA of a specific gene and the onset of the disease. They identified an important molecular signaling pathway in cultured bone marrow cells from patients that is inhibited by the gene methylation. Treating these cells with a drug that inhibits methylation led to reactivation of the gene and the associated signalling pathway that promotes healthy bone formation.Studies of genetic and molecular signaling processes in the bone disease osteonecrosis, when it is associated with steroid use, reveal insights into disease development and suggest new approaches for treatment. Steroid drugs increase the risk of osteonecrosis, in which bone tissue dies due to insufficient blood supply, but the mechanism of this effect is unclear. Researchers in China, led by Zhibo Sun at Wuhan University, investigated a link between the aberrant addition of methyl groups (CH However, none of these can explain the underlying mechanism. Our previous study demonstrated that the abnormal differentiation of bone marrow mesenchymal stem cells (MSCs) is a principal mechanism involved in the onset and progression of this disease11.Since glucocorticoids (GC) are widely used in daily clinical practice, their exogenous usage ranks first among the known risk factors for nontraumatic osteonecrosis of the femoral head, called glucocorticoid-induced osteonecrosis of femoral head (ONFH). Currently, several alternative mechanisms have been postulated for GC-induced ONFH, such as fat embolisation12, cardiocytes13,chondrocytes14, and adipocytes15. In GC-induced ONFH, decreased numbers, hypoproliferative activity and abnormal differentiation of MSCs of the femoral head, neck, and metaphysis result in poor self-repair and a negative prognosis. When the adipocyte population becomes more numerous than the osteoblast population, the intraosseous pressure may rise, ultimately causing ONFH. Therefore, to treat GC-associated ONFH, it is important to inhibit MSCs from undergoing adipogenesis and promote their osteogenesis.MSCs, originally identified in adult bone marrow, can proliferate and differentiate into multiple mesodermal lineages, such as osteoblasts18. This pathway controls both the proliferation and differentiation of osteoblastic precursors and maintains mature osteoblasts20. As a receptor for the Wnt signaling pathway, Frizzled1 (FZD1) plays an important role in osteoblast mineralization. Its promoter is regulated by several transcription factors, including Sp1, E2F1, and AP223. Recent studies showed that FZD1 is closely related to bone mineral density (BMD)25. However, its expression in the MSCs of patients with steroid-induced ONFH has not been investigated yet.The canonical Wnt signaling pathway plays a vital role in the regulation of bone homeostasis, which inhibits the differentiation of MSCs into chondrocytes and adipocytes while promoting osteoblastic differentiationIn this study, we focused on FZD1 in MSCs and investigated whether FZD1-promoter hypermethylation is present in patients with ONFH and results in Wnt/β-catenin signaling inactivation and subsequent cell dysfunction. We performed methylation analysis of the promoter CpG islands of FZD1 by bisulfite sequencing (BSP) and investigated whether demethylation of FZD1 exerts a beneficial influence on cell viability and osteogenic differentiation. We attempted to confirm that FZD1-promoter hypermethylation is related to Wnt/β-catenin signaling inactivation in the development of GC-induced ONFH.26. After written informed consent was obtained from patients, bone marrow aspirates (5 mL) were procured from the proximal ends of femurs while inserting a tapered awl into the femoral canal during hip replacement surgery.This study was approved by the Ethics Committee of Renmin Hospital. All methods were performed in accordance with the relevant guidelines and regulations of the authors’ institution. Between June 2016 and May 2017, 21 patients  with GC-induced ONFH were selected at the authors’ institution , and 22 subjects with femoral neck fractures  were enrolled as controls. Clinical characteristics for all participants are summarized summarized in Table 27. Adherent cells were cultured for 12 to 14 days until they attained a confluence of greater than 80%. The cells were then digested with a solution of 0.25% trypsin and 0.02% EDTA  and replated at a 1:2 dilution for initial subculture. hMSCs underwent this treatment three times before they were collected for further use.hMSCs were isolated from bone marrow aspirates and cultured as described previously3 cells per well28. After adherence to the plates, the initial defining medium was aspirated away and replaced with complete medium supplemented with 5’-Aza-dC  in the treatment group. At 24, 48, and 72 h, cell proliferation was assayed by MTT according to the manufacturer’s instructions.As described previously, MSCs were plated in 96-well plates at a density of 2 × 10Cells were fixed in 4% paraformaldehyde for 24 h, permeated with 0.2% Triton X-100 (Sigma), blocked, and then finally incubated with primary antibodies at a dilution of 1:100 at 4 °C overnight. Cells selected for DKK1, FZD1, and β-catenindetection were incubated with FITC- and Cy3-conjugated secondary antibodies at 37 °C for an additional one hour using standard concentrations from the supplier. Then, the slides were washed and mounted with CitiFluormountant .Cells were washed twice with ice-cold PBS, scraped into 0.2 mL of buffer , and incubated on ice for 20 min, followed by centrifugation at 12,000 rpm for 10 min. Protein concentrations were quantified by a BCA Protein Assay Kit . Afterwards, proteins were diluted to equal concentrations, boiled for 5 min, separated by 10% SDS-PAGE, and then blotted onto polyvinylidene fluoride (PVDF) membranes , which were probed with a FZD1 antibody , β-catenin antibody , Runx2 antibody  and PPARγ antibody  overnight at 4 ℃. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature . GAPDH was used to normalize for protein loading.29. For Oil red O staining, the cells were washed twice with PBS and fixed with 4% formaldehyde in PBS for 30 min at room temperature. Subsequently, they were stained for 1 h at room temperature with filtered Oil red O solution, washed twice with PBS, visualized under light microscopy and photographed. To extract the incorporated Oil red O, 1 mL of isopropanol was added to each well followed by 15 min of shaking at room temperature. After appropriate dilution, the absorbance of triplicate samples was read at 490 nm.Adipogenic and osteogenic differentiation of human MSCs were performed as previously described2) containing chromogen substrate solution composed of 33 μl of 50 mg/ml nitro blue tetrazolium (NBT) and 16.5 μl of 50 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP). Cells were stained with BCIP/NBT substrate for 30 min. Finally, the substrate solution was removed, and the cells were rinsed with deionized water, visualized under light microscopy and photographed.For ALP staining, the cells were washed twice with PBS and fixed with 4% formaldehyde in PBS for 30 min at room temperature. After three washes with PBS, ALP staining was performed by the addition of 5 ml of staining buffer  staining, the cells were washed twice with PBS and fixed with 4% formaldehyde in PBS for 30 min at room temperature. After a brief wash with PBS, they were stained for 20 min with 40 mM ARS solution (pH 4.2). Next, they were rinsed five times with PBS to reduce nonspecific staining. Using Meta Morph imaging software , osteogenic differentiation was quantified by measuring the area stained with Alizarin Red S. Measurements were performed in duplicate for each experiment, and experiments were repeated three times.Total RNA was extracted via standard protocols using standard commercial kits . Real-time PCR was performed using SYBR Green Master Mix according to the protocols of the supplier . The primer sequences are shown in Table 30. Briefly, total genomic DNA was isolated from MSCs using a DNeasy Tissue Kit (Cwbiotech). Two micrograms of genomic DNA were denatured in a volume of 50 μL by freshly prepared 0.3 M NaOH for 30 min at 42 ℃. After denaturation, 30 μL of freshly prepared hydroquinone (10 mM) and 510 μL of sodium bisulfite  were added and incubated at 50 ℃ for 16 h. Modified DNA was purified using a DNeasy spin column (Qiagen) and eluted in a volume of 50 μL. This was followed by desulfonification by adding 5.5 μL of 3 M NaOH for 15 min at 37 ℃. Samples were neutralized by adding 33 μL of ammonium acetate , followed by ethanol precipitation and resuspension in water.Bisulfite conversion was performed as previously describedPCR was performed at 95 ℃ for 5 min followed by 40 cycles of 95 ℃ for 30 s, 55 ℃ for 30 s and 72 ℃ for 1 min with a final extension at 72 ℃ for 7 min. Primer pairs were designed using a custom interface with primer3. The PCR products were tested in 2% agarose gel and then cloned into the pEASY-T1 vector . Colony PCR was undertaken to screen the positive colonies. Clones with correctly sized PCR products were sequenced on an ABI sequencer with dye terminators . Based on the sequencing results for ten clones, the methylation frequency was determined for each CpG site.P < 0.05) were considered statistically significant. All experiments were repeated three or more times.Statistical analysis was carried out via SPSS version 13.0 for Windows . Significant differences were determined using either the Mann-Whitney U test or the Wilcoxon signed-rank test. Data are presented as the mean ± standard deviation. Probabilities lower than 5% . Following treatment with 15 μM 5’-Aza-dC, the cellular viability in the GC-induced ONFH group increased by more than one fourth but was still a little lower than that in the control group at t = 72 h (P < 0.05).As shown in Fig. Compared to the control group, FZD1 and β-catenin showed obviously weak staining and Dkk1showed strong staining in the ONFH group. Following treatment with 5’-Aza-dC, β-catenin and FZD1 staining was obviously increased but was still weaker than that of the control group Fig. . HoweverP < 0.05). Treatment with 15 μM 5’-Aza-dC resulted in weakened staining, which was almost the same level as that of the control group (P > 0.05).As shown in Fig. As shown in Fig. P < 0.05).As shown in Fig. Our results showed that 5’-Aza-dC markedly promoted FZD1, β-catenin and Runx2 translation while inhibiting PPARγ translation. It was observed that MSCs in the ONFH group showed obviously low expression of FZD1 and β-catenin compared to that of the control group. When treated with 5’-Aza-dC for 72 h, this expression correspondingly increased Fig. . Cells iP < 0.01), reaching a level nearly 5-fold higher at 72 h after treatment with 5’-Aza-dC, which was almost the same level as that of the control group (P > 0.05). Meantime, CyclinD1 and Axin2 transcription was elevated to varying degrees by treatment with 5’-Aza-dC. Pretreatment with 5’-Aza-dC also helped to promote RUNX2 and osteocalcin transcription and decrease PPARγ transcription.Our results showed that 15 μM5’-Aza-dC markedly promoted the transcription of FZD1, CyclinD1, Axin2, and osteogenesis-related genes  while inhibiting adipogenesis-related gene (PPARγ) transcription. As shown in Fig. To determine whether the increased expression of FZD1 in MSCs had an underlying epigenetic basis, DNA methylation status in the FZD1 promoter was examined by bisulfite sequencing. A schematic overview of the promoter structure is shown in Fig. 11. However, the exact mechanism has not yet been clarified.Currently, we see an increased incidence of ONFH due to the widespread use of exogenous glucocorticoids. At the present time, the precise mechanism underlying steroid-associated ONFH has not yet been elucidated, although numerous studies have been done. Epigenetics, an effective method to study the interplay between environmental signals and the genome, has received a great deal of recent attention. Our previous studies demonstrated that DNA hypermethylation plays an important role in the pathophysiology of GC-associated ONFH. It is beneficial for dysfunctional MSCs to reverse DNA hypermethylation modifications31. Mutations in components of the Wnt/β-catenin pathway are associated with decreased bone mineral density and increased fracture incidence as well as other skeletal disorders32. Dickkopf-1 (DKK1), one of the Wnt inhibitors, showed increased expression in ONFH patients with histories of corticosteroid and alcohol intake33. Some studies demonstrated that FZD1 is very important in osteoblast differentiation and mineralization, and its promoter can be regulated by several transcription factors23. Our pilot study showed that FZD1 was obviously decreased at the transcription and translation levels in GC-associated ONFH. Therefore, we hypothesized that FZD1 promoter hypermethylation resulted in low expression of FZD1, downregulation of the Wnt/β-catenin signaling pathway and MSC dysfunction in the MSCs of patients with GC-associated ONFH. Reversal of the hypermethylation status of the FZD1 promoter is helpful for reactivation of the Wnt/β-catenin signaling pathway and improvement of MSC osteogenic differentiation.The Wnt/β-catenin signaling pathway is reported to be involved in the pathogenesis of early-stage ONFHFZD1 gene in MSCs from 43 patients with GC-induced ONFH and femoral neck fractures. We found that the methylation ratio of the FZD1 promoter in the GC-induced ONFH group was obviously higher than that in femoral neck fractures group. Additionally, FZD1 mRNA and protein levels in the GC-induced ONFH group were one sixth and one tenth of those in femoral neck fractures group, respectively. Apparently, expression of the FZD1 gene was partly inhibited due to the promoter hypermethylation in the GC-induced ONFH group.In this study, we assessed methylation at CpG islands of the 34. DNA methylation is a key mechanism associated with pluripotency and self-renewal of stem cells. It was reported that 5-Aza induced the direct conversion of adult gingival MSCs into cells of three embryonic lineages, suggesting their potential use for autologous cell therapy35. Cho et al. demonstrated that epigenetic modification induced by 5’-Aza-dC permitted direct programming of adipocytes into osteoblasts in a mouse model of osteoporosis, indicating that 5’-Aza-dC is a promising treatment for osteoporosis36.5′-Aza-dC, an inhibitor of DNA methyltransferase (DNMT), was demonstrated to be involved in cell proliferation and differentiation11 and the recent report from Katarzyna Kornicka et al.37, who found that 5-azacytidine reduced reactive oxygen species accumulation, ameliorated superoxide dismutase activity, increased cellular proliferation and the BCL-2/BAX ratio, and ultimately slowed down and even reversed aged-related degenerative changes in MSCs. Our previous data showed the similar effects of 5-azacytidine on the MSCs of patients with GC-associated ONFH.Notably, we observed that cellular viability in the GC-induced ONFH group was remarkably lower than that in the control group at three different time points. Following treatment with 15 μM 5′-Aza-dC, cellular viability in the ONFH group increased by more than one fourth, while methylation ratios decreased to 1.8% (region 1) and 4.6% (region 2). This finding is consistent with our previous articleFrom an immunofluorescence analysis, we found that the MSCs of patients with GC-associated ONFH demonstrated hampered intranuclear translocation of β-catenin compared to that of the control group. 5’-Aza-dC can promote the intranuclear translocation of β-catenin. FZD1 was weakly expressed in the MSCs of patients with GC-associated ONFH, and this expression was increased by treatment with 5’-Aza-dC. However, expression of the DKK1 protein exhibited an opposite pattern. The PCR results showed that 5’-Aza-dC also increased the mRNA expression of FZD1, Axin 2, Cyclin D1 and Runx2, which were decreased in GC-associated ONFH. Moreover, MSCs treated with 5’-Aza-dC showed weakened adipogenesis and enhanced osteogenesis according to the oil red O, ALP and ARS staining results. Taken together, there is a good possibility that the beneficial effects of 5’-Aza-dC on GC-associated ONFH are attributable to FZD1-promoter demethylation of MSCs and reactivation of the Wnt/β-catenin signaling pathway. However, the underlying mechanism must be studied further.FZD1 gene is present in patients with ONFH, resulting in Wnt/β-catenin signaling inactivation and subsequent cell dysfunction. By comparative observation, we confirmed that 5’-Aza-dC, at a suitable concentration, benefits the MSCs of patients with GC-associated ONFH by inducing de novo FZD1 expression. This study has provided new information regarding treatment targeting epigenetic changes in ONFH. However, more specific in vivo studies are needed to further elucidate the molecular pathomechanism underlying the development of ONFH.In summary, this study has demonstrated that aberrant CpG island hypermethylation of the