Axin is a key scaffolding protein responsible for the formation of the β-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of cellular concentration of Axin protein has a profound effect on Wnt/β-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, the deubiquitinase responsible for Axin deubiquitination and stabilization remains unknown. Here, we identify USP7 as a potent negative regulator of Wnt/β-catenin signaling through CRISPR screens. Genetic ablation or pharmacological inhibition of USP7 robustly increases Wnt/β-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain, and promotes deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblast differentiation and adipocyte differentiation through increasing Wnt/β-catenin signaling. Our study reveals a critical mechanism that prevents excessive degradation of Axin and identifies USP7 as a target for sensitizing cells to Wnt/β-catenin signaling. Axin is a scaffolding protein known for its role in Wnt signalling that can be marked with a variety of post-translational modifications. Here, Cong et al. demonstrate that USP7 de-ubiquinates Axin and that canonical Wnt signaling output can be increased with USP7 inhibitors. The Wnt/β-catenin pathway regulates proteolysis of transcription cofactor β-catenin. In the absence of Wnt, β-catenin is bound to the β-catenin destruction complex, which contains the scaffolding protein Axin, the tumor suppressor adenomatous polyposis coli (APC), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α). In this complex, β-catenin is phosphorylated by CK1α and GSK3, and targeted for ubiquitin-dependent proteasomal degradation. In the presence of Wnt, Wnt ligand binds to the Frizzled receptor and the LRP5/6 co-receptor and induces phosphorylation of LRP5/6, which leads to decreased activity of the β-catenin destruction complex and stabilization of β-catenin. Accumulated β-catenin enters the nucleus, binds to TCF family transcription factors, and enhances the transcription of β-catenin target genes. Wnt/β-catenin signaling plays important roles in many important biological processes. For instance, Wnt/β-catenin signaling plays a pivotal role in the mesenchymal stem cell (MSC) lineage specification and early differentiation5. Wnt/β-catenin signaling drives osteoblast differentiation of mesenchymal stem cells6, while suppresses the adipogenic program7. Aberrant Wnt/β-catenin signaling has been associated with many human diseases, such as degenerative diseases and cancer2.The Wnt/β-catenin signaling pathway is an evolutionarily conserved signaling pathway that plays essential roles in embryonic development and adult tissue homeostasis8, and E3 RNF146 mediates PARsylation-dependent ubiquitination and degradation of Axin9. Tankyrase inhibitors strongly inhibit Wnt signaling through stabilizing Axin10. In addition, E3 SIAH binds to the GSK3-binding domain of Axin and mediates Wnt-induced degradation of Axin11, which serves as a positive feedback mechanism to amplify Wnt/β-catenin signaling. Although mechanisms that promote Axin degradation and their importance in Wnt/β-catenin signaling are established, the deubiquitinase that removes polyubiquitin chain from Axin to prevent its excessive degradation has been mysterious. Significantly, since Wnt/β-catenin signaling is exquisitely sensitive to the concentration of Axin, small molecules blocking deubiquitination of Axin are expected to sensitize cells to Wnt signaling and might have therapeutic values.As a key scaffolding protein, Axin directly interacts with β-catenin, GSK3, and APC, and is directly responsible for the formation of the β-catenin destruction complex. Since the concentration of Axin is correlated with the concentration of the β-catenin destruction complex, mechanisms that control the protein level of Axin have major impacts on Wnt/β-catenin signaling. Through genetic and chemical genetic screens, we have previously identified two independent E3 ubiquitin systems that promote Axin degradation. Tankyrase binds to the N-terminus of Axin and promotes PARsylation of Axin12, MDM214, HDMX14, N-MYC15, PTEN16, FOXP317, Gli18, Tip6019, and ERCC620. Through its deubiquitination activity, USP7 regulates the stability, function, or localization of its substrates, and plays important roles in cancer development, cell signaling, DNA damage repair, epigenetic regulation, and immune responses (reviewed by21).Ubiquitination is an enzymatic process by which proteins are modified with ubiquitin chains and this process can be reversed by deubiquitinases (DUBs). The process of ubiquitination and deubiquitination is extremely dynamic and involves specific protein-protein interactions. USP7 , is an ubiquitin hydrolyzing enzyme belonging to the ubiquitin specific protease (USP) family. USP7 has a diverse array of substrates, such as p53Here, we identify USP7 as a potent negative regulator of Wnt/β-catenin signaling. Mechanistically, USP7 directly interacts with Axin through its N-terminal TRAF domain and stabilizes Axin by erasing the poly-ubiquitination modification. Genetic or pharmacological inhibition of USP7 promotes osteoblast differentiation and attenuates adipocyte differentiation through activation of Wnt signaling. Our work reveals USP7 as a bona fide deubiquitinase for Axin and demonstrates its negative role in Wnt/β-catenin signal transduction.CSNK1A1, ZNRF3, APC, and AXIN1) of Wnt signaling scored clearly in this screen , a transcription reporter with multiple TCF binding sites. STF-GFP reporter cells were mutagenized by a pooled lenti-viral guide RNA (gRNA) library, treated with Wnt3a conditioned medium (CM) and subjected to FACS analysis. Cells expressing low GFP signal and high GFP signal were collected and subjected to next generation sequencing (NGS) and informatics analysis  cells stably expressing empty vector, wild-type USP7 (WT), or the USP7 catalytic dead mutant C223Aion Fig. . In contion Fig. , presumaion Fig. . Combini28. Because of this important function of USP7, significant efforts have been made to develop small molecule USP7 inhibitors to treat cancers with wild-type p53. However, early USP7 inhibitors have low potency/selectivity29. Although these inhibitors stabilize p53 protein, it is still not clear whether their cellular toxicity is solely mediated by USP7 inhibition. Recently, USP7 inhibitors with significantly improved potency and selectivity have been reported. We found that the new generation of USP7 inhibitors Almac430, FT67129, and P5042931 strongly increased STF-GFP in parental HEK293T cells, but not in HEK293T USP7 knockout cells  assay in a dose response manner   mutant  domain, a middle catalytic domain and a carboxyl-terminal tandem UBL domain (TUD), which contains five ubiquitin-like folds41 Fig. . Both thant Fig. . In a reant Fig. . In addiant Fig. . These r24. To rule out the possibility that the interaction between USP7 and Axin is mediated by β-catenin, we included β-catenin as a control in USP7 co-immunoprecipitation assay. Consistent with the pull down data, Flag-Axin1strongly interacted with ectopically expressed wild-type USP7, but not the WA mutant  and β-catenin target gene Axin2 and Lef1 in C3H10T1/2 and ST2 cells . ODM increased the level of active β-catenin in C3H10T1/2 cells, which is further enhanced by USP7 inhibitor Almac4, and this effect is abolished by porcupine inhibitor WNT974 Fig. . In the nes Fig. , which wnes Fig. . Taken t48. We investigated the role of USP7 in adipocyte differentiation using mouse preadipocyte cell line 3T3-L149. Unlike C3H10T1/2 and ST2, proliferation of 3T3-L1 is not affected by USP7 inhibition although 3T3-L1 expresses wild-type p5350. Knockout Usp7 using independent gRNAs enhanced Wnt3a-induced accumulation of active β-catenin , HCT116 (β-catenin mutant), and Ls174t (β-catenin mutant) without inhibiting β-catenin signaling in these cells  cells43, four in MDM259, two in HDMX46, four in Gli18. We have identified three binding sites of USP7 in Axin1, two N-terminal to RGS domain and one between RGS domain and GSK3-binding domain. It is possible that multiple interaction sites can enhance USP7 interaction or confer more dynamic regulation. Of note, 72PEGS75 of a.a. 63–87, which corresponds to the consensus USP7 binding sequence, falls into the second tankyrase binding motif of Axin160. Whether and how USP7-mediated Axin deubiquitination is regulated should be addressed in future studies. In addition, although our data suggest Axin as a major target of USP7 for Wnt regulation, it is possible that regulation of other proteins also contributes to the inhibitory activity of USP7 in Wnt signaling.It is still not clear whether USP7-mediated deubiquitination serves as a general buffer system to dampen Axin degradation, or it is regulated in a more dynamic fashion. Wnt-induced Axin ubiquitination is prolonged in USP7 knockout cells Fig. , suggestAlthough many small molecule Wnt inhibitors have been identified, few small molecule sensitizers of Wnt/β-catenin signaling exist. USP7 strongly inhibits Wnt/β-catenin signaling through deubiquitination and stabilization of Axin. To our knowledge, USP7 inhibitors represent the most potent agents sensitizing cells to β-catenin signaling beyond GSK3 inhibitors. Selective USP7 inhibitors can potentially be tested in certain disease indications where activation of Wnt signaling is beneficial.HEK293T, Huh7, RKO, U2OS, 3T3-L1, C3H10T1/2 clone 8, HCT116, Ls174t, SW480, HT-29, DLD1, and Caco-2 were purchased from American Type Culture Collection (ATCC), YAPC and ST2 were obtained from DSMZ. HEK293T, YAPC, Huh7, SW480 and 3T3-L1 were grown in DMEM  with 10% FBS  and penicillin/streptomycin . ST2 and DLD1 were maintained in RPMI-1640  with 10% FBS and penicillin/streptomycin. U2OS, HT-29 and HCT116 were grown in McCoy’s 5A  with 10% FBS and penicillin/streptomycin. RKO, Caco-2, Ls174t and C3H10T1/2 were maintained in MEM  with 10% FBS and penicillin/streptomycin. All cultures were grown at 37 °C in a 5% CO2 incubator. Cell lines were tested to be free of mycoplasma contamination, and were not subjected to extra authentication steps.Plasmids were generated using standard recombinant DNA techniques. Site-specific mutagenesis was performed using Q5 Site-Directed Mutagenesis Kit (New England Biolabs). Primers used for cloning are shown in Supplementary Table USP7-g1 (5′CTACGTCGGCTTAAAGAATC3′), USP7-g2 (5′ GATTTCGCACAAAACACGGA3′), CTNNB1-g1 (5′ACAAAACTGCTAAATGACG3′), SIAH1 (5′ TAATGCTGTAGCAGTCTGA3′), AXIN1 (5′ TGAGAAAACCTCTGGTCGTGT3′), AXIN2 (5′ ATACCCTTTTCTCTCTCCCCAC3′), mouse Usp7-g1 (5′ AGACACCAGTTGGCGCTCCG3′), and mouse Usp7-g2 (5′ GGCAGATTTCGCACAAAACA3′).For CRISPR knockout, cells stably expressing Cas9 were infected with lentivirus expressing indicated gRNA and selected by antibiotics. Unless specifically indicated, pool of knockout cells were used for experiments to avoid clonal variation. All gRNA targeting sequences are listed below: Ctrl (5′ GACCGGAACGATCTCGCGTA3′), P-value between the GFP-high and the GFP-low samples. A robust z-score for each gRNA was calculated using the median and mean-absolute deviation across the log2 fold changes. To summarize the results at the gene level, the gRNAs are ranked by the robust z-score, and the statistical significances for each gene enriched toward higher rank (RSA up) were evaluated using the Redundant siRNA Activity (RSA) algorithm. The RSA P-value is shown along with the log2 fold change of the five5 gRNAs per gene for the indicated set of genes.We designed five gRNAs against each gene using Illumina Human BodyMap 2.0 and NCBI CCDS data sets. The gRNA library containing 90,000 gRNAs was synthesized using array synthesis and cloned into a lentivirus vector by Cellecta Inc. STF-GFP reporter cells were transduced at MOI 0.5. Fourteen days post virus transduction, cells were treated with 5% Wnt3a CM overnight, and GFP-high and GFP-low cells were sorted using BD FACSAria Cell Sorter. Genomic DNA was collected and subjected to Illumina DNA sequencing for barcode counts. Raw sequencing reads were aligned to the appropriate library using Bowtie, allowing for no mismatches, and counts were generated. The R software package DESeq2 was used to evaluate differential gRNA representation in the form of log2 fold change and Cells were harvested using cell dissociation buffer  and resuspended in FACS buffer , then subjected to Cytoflex cytometer (Beckman Coulter Life Sciences). FITC channel was used to collect GFP signal, and raw data was analyzed by FlowJo version 10.GUSB as an internal normalization control. The Taqman probes used in this study were obtained from Life Technologies, GUSB (Hs00939627_m1), AXIN1 (Hs00394718_m1), AXIN2 (Hs00610344_m1), LEF1 (Hs01547250_m1), CTNNB1 (Hs00355045_m1), RNF146 (Hs00258475_s1), Gusb (Mm01197698_m1), Axin2 (Mm00443610_m1), Lef1 (Mm00550265_m1), Alpl (Mm00475834_m1), Pparg (Mm00440940_m1), Fap4 (Mm00445878_m1), and Lpl (Mm00434764_m1).Total RNA was extracted by using RNeasy Plus mini kit  and reverse transcribed by TaqMan reverse transcription reagents  according to the manufacturer’s instructions. Gene expression levels were detected by TaqMan probes, and all the experiments were performed in quadruplicate. The comparative cycle threshold method was employed to analysis the gene expression with housekeeping gene ×g for 15 min at 4 °C. Equal amount of proteins were resolved by SDS-PAGE, transferred to nitrocellulose membrane and probed with indicated antibodies.For immunoblotting, total cell lysates were prepared by directly lysing cells with NP40 lysis buffer , followed by centrifugation at 15,000The antibodies used are as follows: β-catenin , Tubulin , APC , USP7 , active β-catenin , HA , Flag , Myc-tag , GFP , Axin1 , GSK3β , CK1 ,and GST .https://imagej.nih.gov/ij/), which measures the integrated density of bands corrected for background. The intensity of target protein and Tubulin bands were quantified by ImageJ, and quantification of normalized band intensity was listed right below the target protein. Uncropped version of immunoblots are shown in Supplementary Figures and Source data file.Immunoblots were quantified using the ImageJ program  or USP7 primary antibody  overnight, and then incubated with magnetic Protein A beads  for 2 h at 4 °C. For Myc immunoprecipitation, cell lysates were directly incubated with Anti-Myc tag magnetic beads  for 2 h at 4 °C. Then Beads were washed three times with lysis buffer, and bound proteins were eluted in SDS sample buffer for immunoblotting analysis.Cells were harvested in cold PBS, the cell pellet was resuspended in hypertonic buffer (10 mM Tris-HCl pH 7.5 and 10 mM KCl), and followed by freeze-thaw for four times. Cleared supernatants were collected after full speed centrifugation. Equal amount of proteins were resolved by SDS-PAGE and probed with β-catenin or active β-catenin antibody.Plasmid transfection was done using FuGene6 (Promega) or Lipofectamine 2000 (Thermo Fisher Scientific) and siRNA transfection was done using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to manufacturer’s instructions. Lentiviruses were produced using HEK293T cells following the standard virus packaging protocol, and used to generate stable cell lines.In vitro purified GST, GST-TRAF, GST-TRAF W165A or GST-TUD was incubated with Flag-Axin1 cell lysate with 20 μl of GSH sepharose 4B resin  for 2hrs at 4 °C in binding buffer . The beads were then washed with binding buffer three times.The same amount of purified His-SUMO tag proteins were incubated with purified GST-TRAF or GST-TRAF W165A with 20 μl Ni-NTA resin  for 1 hr at 4 °C in binding buffer . The beads were then washed with binding buffer three times. The proteins were resolved on SDS-PAGE and examined by coomassie staining.For in vivo ubiquitination assay, HEK293T cells were transfected with plasmids encoding HA-tagged ubiquitin, Myc-tagged Axin1 with control vector, wild-type USP7 or C223A mutant. Two days post transfection, cells were treated with 20 μM MG132 for 6hrs and lysed in 100 μl denature lysis buffer . Cell lysates were boiled for 10 min, diluted 10 times in lysis buffer without SDS, and subjected to immunoprecipitation using HA antibody-conjugated beads. Immunoprecipitates were resolved by SDS-PAGE.For in vitro debuiquitination assay, HEK293T cells co-expressing HA-Ub and Flag-tagged Axin1 or Flag-tagged β-catenin were treated with 10 μM MG132 overnight. Polyubiquitinated Axin1 or β-catenin was purified using Flag-M2 antibody-conjugated beads, then the same amount of beads were incubated with or without 10 ng/µl recombinant full length USP7  in the deubiquitination reaction buffer  at 37 °C for 1 h. The reaction was terminated by the addition of SDS sample buffer. The poly-ubiquitination status of Axin1 and β-catenin was analyzed by HA blotting.In vivo poly-ubiquitination of AXIN1 was evaluated by the TUBE assay. The poly-ubiquitination of target protein was enriched by 20 μM of MG132 treatment. Briefly, equal amount of cell lysates were incubated with agarose-TUBE beads  for 2 h at 4 °C, then the beads were washed three times with lysis buffer, and bound proteins were eluted in SDS sample buffer for immunoblotting analysis.HEK293T cells stably expressing the STF-Luc reporter were pretreated with increasing amounts of USP7 inhibitors, following by overnight Wnt3a CM stimulation. The STF-reporter activity was examined by Bright-Glo assay (Promega). Both HEK293T and HEK293T USP7 KO cells were treated with increasing amounts of USP7 inhibitors and viability of cells was assessed after 48 hrs by using CTG assay (Promega).P5091, MG132 and Bortezomib were purchased from SelleckChem. P22077 and HBX41108 were obtained from TOCRIS. MLN7243 was purchased from Chemietek. Bafilomycin A1 was obtained from MilliporeSigma. The rest of known USP7 inhibitors, WNT974, and TNKS656 were synthesized according to published literature procedures by Novartis.Briefly, C3H10T1/2 and ST2 cells were plated in normal culture medium (MEM and 10% FBS for C3H10T1/2 and RPMI1640 and 10% FBS for ST2 cells), and allowed to adhere overnight. On the following day, culture medium was replaced with osteoblast differentiation medium . The medium was changed every other day for 12 days.To determine alkaline phosphatase enzymatic activity, cells were fixed for 10 min with 10% formalin in PBS and incubated with NBT/BCIP substrate solution  for 30 min.The 3T3-L1 cells were cultured in DMEM with 10% FBS. Adipocyte differentiation was induced post cells growing to confluency by replacing the culture medium containing 1 µM Dexamethasone, 500 µM IBMX, 1 µg/ml insulin for 2 days. On day 3, medium was changed to the culture medium supplemented with insulin (1 µg/ml) and left for 7 days (medium was changed every other day). The lipid droplets were analyzed by Oil-Red O staining .Colorectal cancer cells were seeded in six-well plates at low density  and allowed to attach for overnight. DMSO or Amac4 was added directly into each well at indicated concentration. The medium was refreshed every three days with Almac4 at indicated concentration. The plates were washed with PBS and stained with crystal violet when DMSO-treated wells reached confluency.Further information on research design is available in the Supplementary InformationSupplementary datasetReporting SummarySource Data


Protein arginine methyltransferases (PRMTs) catalyze the methylation of arginine residues on both histones and non-histone proteins. PRMT5, a member of the PRMT family, is overexpressed in a wide variety of cancers and its activity is associated with cell transformation. Moreover, its expression is associated with a decrease in patient survival in several cancers, a rationale for developing highly potent inhibitors of its enzymatic activity. However, most studies do not take into account the subcellular localization of PRMT5, which can modify its properties. Indeed, our team recently showed that PRMT5 nuclear expression is associated with prolonged survival. These results corroborated findings in prostate cancer, in which the nuclear fraction of PRMT5 was responsible for inhibiting cell growth, while the cytoplasmic fraction promoted cell growth. In conclusion, this criterion should be evaluated prior to administering PRMT5 inhibitors, which may have adverse effects. Protein arginine methyltransferases (PRMTs) catalyze the methylation of arginine residues on a variety of proteins including histones and non-histone proteins. Arginine methylation is a frequent post-translational modification involved in various cellular processes, such as DNA transcription, mRNA splicing, signal transduction, protein interaction and subcellular protein localization , 2. MembIn this context, most studies have highlighted that PRMT5 overexpression is associated with a decrease in patient survival and an increase in ovarian, lung, multiple myeloma and breast tumor growth -7 Figur, 1B. FurLKB1, since its nuclear expression has been associated with increased survival in breast cancer, whereas its cytoplasmic expression is associated with a decrease in survival [However, we would like to raise awareness of the dangers of indiscriminately using such inhibitors for different types of cancers. Indeed, database analysis highlighted that the expression of PRMT5 can also be associated with prolonged survival for instance in gastric cancers , at the survival . More resurvival , and by survival . These fsurvival . Overall these observations emphasize the importance of PRMT5 localization in regulating its activity and its oncogenic properties. This criterion should be evaluated prior to administration of PRMT5 inhibitors, which may thus have adverse effects. It would be of interest to understand the mechanisms involved in PRMT5 regulation in the nucleus of tumoral cells and to investigate whether its dual prognostic value can be extended to other cancers.


Adherens junctions, consisting of cadherins and catenins, are a group of cell-to-cell junctions that mediate mechanistic linkage between neighboring cells. By doing so, adherens junctions ensure direct intercellular contact and play an indispensable role in maintaining tissue architecture. Considering these critical functions, it is not surprising that adherens junctions are frequently involved in disease. In the present study, the effects of bile duct ligation—a surgical procedure to experimentally induce cholestatic and fibrotic liver pathology—on hepatic adherens junctions were investigated in mice. In essence, it was found that liver mRNA and protein levels of E-cadherin, β-catenin and γ-catenin drastically increase following bile duct ligation. These results could suggest a cytoprotective role for hepatic adherens junctions following bile duct ligation. Adherens junctions form bridges that connect the cytoskeleton of adjacent cells and hence are key structures in the maintenance of cellular architecture . They arGiven the critical roles of adherens junctions and their constituents in cellular homeostasis, it is not surprising that these structures are frequently involved in disease. In this respect, β-catenin levels steeply increase in acetaminophen-induced acute liver failure in mice . LikewisThe present study was initiated in order to investigate the effects of bile duct ligation (BDL), a well-known surgical procedure that triggers cholestatic and fibrotic insults, on hepatic adherens junctions in mice. Focus is put on monitoring changes in the expression and cellular localization of E-cadherin, β-catenin and γ-catenin.g, and serum was stored at −20 °C. Livers were excised and fragments were fixed in 10% phosphate-buffered formalin or snap-frozen in liquid nitrogen with storage at −80 °C.Male C57BL/6 mice were obtained from Jackson Laboratories (USA). Animals were housed in the animal facility of the School of Veterinary Medicine and Animal Science of the University of São Paulo. Mice were kept in a room with ventilation , relative humidity , controlled temperature  and light/dark cycle 12:12 and were given water and balanced diet  ad libitum. This study has been approved by the Committee on Bioethics of FMVZ-USP (protocol number 9999100314) and all animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals”. The BDL or sham models were set up in 8-weeks to 12-weeks-old mice as described elsewhere ,16. MiceSerum levels of alanine aminotransferase , aspartate aminotransferase , alkaline phosphatase , conjugated and total bilirubin (mg/dL) were measured with an automated spectrophotometric Labmax 240 analyzer  after appropriate dilution of samples.For microscopic evaluation, formalin-fixed liver fragments were embedded in paraffin and 5 µm sections were stained with Sirius red for blinded evaluation of the liver as previously described . Morphom(−ΔΔCq) formula [Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis of liver tissue was assayed as explained elsewhere . The Taq formula .Immunoblot analysis of liver tissue was performed as previously described  with sli® . Then, 5 µm liver sections were fixed in methanol for 15 min at −20 °C and blocked in blocking buffer based on 5% w/v donkey serum  during 1 h. Subsequently, liver sections were incubated with primary antibodies directed against E-cadherin, β-catenin and γ-catenin  in blocking buffer based on 5% w/v donkey serum  overnight at 4 °C. Primary antibodies used for immunohistochemistry analysis are presented in ® 488-conjugated secondary antibody  in blocking buffer based on 5% w/v donkey serum . Thereafter, Vectashield with 4′,6-diamidino-2-phenylindol  was used to stain nuclei and as mounting medium. Detection was performed using a fluorescence microscope Nikon Eclipse Ti .Flash frozen liver tissue samples were embedded in Tissue Freezing Mediumn) or Shapiro–Wilk normality test for low n) using GraphPad Prism7 software , with probability (p) values of less than or equal to 0.05 considered as significant.All data were expressed as mean with data range . Results were statistically processed by 2-tailed unpaired student t-tests and Welch’s correction or Mann–Whitney tests depending on the distribution  in serum levels of the general liver injury markers ALT and AST following 20 days of BDL. This coincided with elevated (p ≤ 0.0001) quantities of the cholestatic indicators ALP, conjugated and total bilirubin (p ≤ 0.0001) normalized collagen area ratio in liver tissue of BDL-subjected mice compared to sham-operated counterparts  in mRNA quantities of liver E-cadherin, β-catenin and γ-catenin (p ≤ 0.0001), β-catenin (p ≤ 0.01) and γ-catenin (p ≤ 0.0001) (p ≤ 0.0001)  . Besides 0.0001) . The enh 0.0001) .As such, BDL is known to differentially affect cell-to-cell junctions in liver, including tight junctions , desmosoOn the other hand, the BDL-associated alteration of hepatic adherens junction components might equally affect cellular signaling in hepatocytes. As observed in the present study, BDL treatment elevates total β-catenin protein levels in liver. This could lead to activation of the Wnt pathway and enhanced expression of target genes, in turn promoting proliferation . This alAdditionally, liver APC protein expression levels were investigated. Immunoblot analysis showed that the BDL model causes an increase in truncated APC. APC mediates intercellular adhesion by targeting β-catenin and γ-catenin for degradation. Therefore, APC proteins are regulators of catenin levels and are described as components of the canonical Wnt pathway ,47. In tThe relevance of increased expression of adherens junction building stones upon BDL remains to be elucidated. Enhanced hepatic E-cadherin production following BDL in mice was also observed by other groups . Further


FABP2 and PLIN2, in these cells. Conversely, activation of Wnt/β-catenin signaling decreased protein and mRNA levels of HMGCS2, βHB production, and expression of PPARγ and its target genes in LS174T and Caco2 cells and mouse intestinal organoids. Moreover, inhibition of PPARγ reduced HMGCS2 expression and βHB production, while activation of PPARγ increased HMGCS2 expression and βHB synthesis. Furthermore, PPARγ bound the promoter of HMGCS2 and this binding was enhanced by β-catenin knockdown. Finally, we showed that HMGCS2 inhibited, while Wnt/β-catenin stimulated, glycolysis, which contributed to regulation of intestinal cell differentiation. Our results identified HMGCS2 as a downstream target of Wnt/β-catenin/PPARγ signaling in intestinal epithelial cells. Moreover, our findings suggest that Wnt/β-catenin/PPARγ signaling regulates intestinal cell differentiation, at least in part, through regulation of ketogenesis.The Wnt/β-catenin pathway plays a crucial role in development and renewal of the intestinal epithelium. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting ketogenic enzyme in the synthesis of ketone body β-hydroxybutyrate (βHB), contributes to the regulation of intestinal cell differentiation. Here, we have shown that HMGCS2 is a novel target of Wnt/β-catenin/PPARγ signaling in intestinal epithelial cancer cell lines and normal intestinal organoids. Inhibition of the Wnt/β-catenin pathway resulted in increased protein and mRNA expression of HMGCS2 and βHB production in human colon cancer cell lines LS174T and Caco2. In addition, Wnt inhibition increased expression of PPARγ and its target genes,  Mammalian intestinal epithelium is maintained by a continuous renewal process comprised of proliferation, migration, differentiation, and apoptosis ,2. A varThe Wnt/β-catenin pathway plays an essential role in early development and tissue maintenance of adults through regulating T-cell factor/lymphoid enhancer-binding, factor-dependent transcription of its target genes ,9. Wnt/βPeroxisome proliferator-activated receptors (PPARs), which function as transcription factors, are a group of ligand-activated nuclear receptors consisting of three subtypes of PPARs  ,15. PPARHMGCS2 is the rate-limiting enzyme for ketogenesis, which leads to the production of ketone bodies including β-hydroxybutyrate (βHB) . ExpressIn this study, we showed that Wnt/β-catenin/PPARγ signaling regulates HMGCS2 expression and βHB production in intestinal cells. Moreover, we found the inhibitory role of the HMGCS2 in glycolysis, which regulates intestinal cell differentiation. Dysregulated Wnt/β-catenin signaling may result in aberrant regulation of ketogenesis and an imbalance in proliferation and differentiation patterns within the intestinal crypts, which is associated with a number of intestinal pathologies.2 incubator. Authentication and mycoplasma contamination tests were performed as described previously [d-glucose (2-DG), a glucose analog that inhibits glycolysis, was obtained from Sigma-Aldrich . Transfection with ON-TARGETplus SMARTpool siRNAs for NTC , β-catenin , c-Myc , PPARγ , and HMGCS2   was performed using Lipofectamine RNAiMAX  according to the manufacturer’s protocol. Adenoviruses encoding GFP  and human c-Myc (Ad-c-Myc) were purchased from Vector BioLabs .The human colon cancer cell lines used in these studies, LS174T and Caco2, were obtained from the American Type Culture Collection . LS174T and Caco2 cells were cultured in EMEM supplemented with 10% FBS and in MEM with 15% FBS, 1% sodium pyruvate, and 1% nonessential amino acids. The cells were maintained at 37 °C in a humidified 5% COeviously . Recombieviously , were obWestern blotting was carried out as described previously . The antIsolation and culture of mouse small intestinal organoids was carried out as described previously . BrieflyHMGCS2 , PPARγ , PPARα , FABP2 , PLIN2 , IAP , SI , Waf1p21 , CDX2 , and GAPDH  according to instruction provided by the manufacturer (Applied Biosystems).Total RNA isolation and RT-PCR analysis were performed as described previously . BrieflyHMGCS2 promoter region containing PPRE was amplified using the primers: forward, 5′-CAGCCATTCCCACACATGCTCA-3′, and reverse, 5′-GACTTTATAAAGCCCCAAGACT-3′. The primers for the distal region of the IFN-λ1 promoter as non-regulated control: forward, 5′-TTTAAGGGCAGGTGCAGGGTGTC-3′, and reverse, 5′-TTACCCAATGTGGTGGGCACCATC-3′ [Chromatin Immunoprecipitation (ChIP) Assay Kit was obtained from Millipore . ChIP assay was performed using an anti-PPARγ antibody or IgG (Santa Cruz Biotechnology) in accordance with the manufacturer’s protocol (Millipore). LS174T cells were transfected with NTC or β-catenin siRNA, and DNA for the LS174T cells was isolated and analyzed by ChIP-qPCR using PerfeCTa SYBR Green SuperMix, ROX . The sequence from the CCATC-3′ . ChIP efCCATC-3′ ,36.Intracellular βHB concentration was determined using a Beta-Hydroxybutyrate Assay Kit  according to the manufacturer’s protocol. Each plotted value was normalized to cell number used from cell lines and total amount of protein used from organoid cultures, respectively.The DNA-binding activity of PPARγ or PPARα was assessed using PPARγ or PPARα Transcription Factor Assay Kits (Abcam), respectively, according to manufacturer’s instruction. Briefly, the nuclear proteins, extracted using a Nuclear Extraction Kit (Abcam), was added in wells immobilized with specific PPRE sequences. After incubation with the primary anti-PPARs antibody and HRP-conjugated secondary antibody subsequently, the absorbance was measured at 450 nm to determine the transcriptional activity of PPARγ or PPARα.4 cells/well in a XF96 plate 24 h before the measurement. The glycolysis stress test was performed according to manufacturer’s protocol and the measurements were normalized to the protein contents in each well. The relative levels of glycolysis and glycolytic capacity, were calculated based on ECAR data obtained in the glycolysis stress tests, using Seahorse Wave software for XF analyzers.The Seahorse XF96 Extracellular Flux Analyzer  was used by the Redox Metabolism Shared Resource Facility of the University of Kentucky Markey Cancer Center to measure extracellular acidification rate (ECAR) for glycolysis of LS174T cells. The cells transfected with siRNA were seeded at the density of 3 × 10Cells were treated with 0, 2.5, 5, and 10 mM 2-DG for the indicated time and intestinal alkaline phosphatase (IAP) activity was determined using Alkaline Phosphatase Yellow (pNPP) Liquid Substrate System  as we have described previously .p-values < 0.05 were considered statistically significant.Bar graphs were generated to represent mean ± SD for each cell culture condition. Relative levels of mRNA, βHB concentration, and transcriptional activity of PPARs and IAP activity were calculated based on mean levels in the NTC group or mean of control cell culture conditions. Fold-changes of western blot densitometry relative to control were calculated for each replicate. Statistical tests were performed using two-sample t-test for relative values and glycolysis ECAR levels, one-sample t-test for western blots, or analysis of variance with contrast statements for pairwise testing or test for linear trend across dose levels. Multiple testing was adjusted using the Holm’s method. HMGCS2 mRNA in these cells, as detected by real time RT-PCR  for 3 days. As shown in HMGCS2 plays a pivotal role in the synthesis of ketone bodies . To detePreviously, it has been reported that HMGCS2 is a target of c-Myc, which suppresses HMGCS2 transcriptional activity , suggestFABP2 (fatty acid binding protein 2) and PLIN2 (perilipin 2 or adipophilin), two PPARγ target genes [FABP2 and PLIN2 in LS174T cells  in LS174T cells. These findings suggest that Wnt/β-catenin signaling regulates HMGCS2 expression through controlling PPARγ expression and recruitment to the HMGCS2 promoter.To further demonstrate the transcriptional regulation of We have demonstrated the regulation of HMGCS2 expression by Wnt/β-catenin/PPARγ signaling pathway. To determine whether this regulation was correlated with the control of ketone body synthesis, we measured the concentration of βHB. As shown in Waf1p21, which suppresses intestinal cell growth and promotes differentiation [Caudal-related homeobox transcription factor 2 (CDX2), which regulates intestinal epithelium renewal [Emerging evidence indicates that the Wnt/β-catenin pathway controls cellular metabolism, including glycolysis ,46,47. Intiation , and Cau renewal , in CacoHMGCS2 promoter. In addition, knockdown of HMGCS2 increases glycolysis, which regulates differentiation in intestinal cancer cells. Taken together, our results suggest a negative regulation of ketogenesis by Wnt/β-catenin signaling.The Wnt/β-catenin pathway plays a critical role in an extensive range of physiological and pathological processes ,9,52. InSox9, which suppresses differentiation associated genes such as CDX2 and Mucin2, is also regulated by the Wnt/β-catenin pathway [Our findings identified HMGCS2 as a novel downstream target gene of Wnt/β-catenin signaling in intestinal cells. A variety of signaling molecules such as Wnt, which is expressed in a gradient along the crypt–villus axis, spatially and temporally regulate a homeostatic balance between proliferation and differentiation in the intestinal epithelium ,4. Among pathway . Moreove pathway . In cont pathway . Based oRecently, we have reported a contributory role of HMGCS2 in intestinal cell differentiation . IncreasHMGCS2 has been identified as a c-Myc target gene, which transcriptionally inhibits HMGCS2 expression in CRC cells [We showed that Wnt/β-catenin signaling regulates ketogenesis independently of c-Myc. Oncogenic c-Myc has been shown to inhibit HMGCS2 expression in CRC cells . The c-MRC cells , it is uOur findings showed that the Wnt/β-catenin pathway regulates ketogenesis by targeting PPARγ. PPARs are ligand-activated transcription factors, which regulate important processes in cellular homeostasis such as cell proliferation, differentiation, and metabolism ,15. AmonMin mice and in SW480 human colon cancer cells [Our results demonstrated that Wnt/β-catenin signaling inhibits HMGCS2 expression by suppressing the expression and activation of PPARγ. In agreement with our findings, it has been shown that activity of Wnt/β-catenin pathway presented negative regulation of PPARγ expression and activation in various pathological conditions, including CRCs ,66,67. Her cells . These cThe results presented here also show that HMGCS2 negatively regulates glycolysis, and inhibition of glycolysis results in increased enterocyte differentiation. The Wnt/β-catenin pathway promotes cell proliferation by altering glycolysis in CRC ,47. InhiIn summary, we showed that HMGCS2 expression and βHB concentration are regulated by the Wnt/β-catenin/PPARγ pathway in human intestinal cancer cell lines and mouse intestinal organoid cultures. Furthermore, our results suggest that HMGCS2 regulates differentiation through regulation of glycolysis. Our findings identified a functional link between Wnt/β-catenin/PPARγ and ketogenesis in intestinal cells D, sugges


Rhodiola rosea L., a traditional Chinese medicine, with multiple biological activities. It has been shown that salidroside possesses protective effects for alleviating diabetic renal dysfunction, contrast‐induced‐nephropathy and other kidney diseases. However, the involved molecular mechanism was still not understood well. Herein, we examined the protective effects of salidroside in mice with Adriamycin (ADR)‐induced nephropathy and the underlying molecular mechanism. The results showed that salidroside treatment ameliorates proteinuria; improves expressions of nephrin and podocin; and reduces kidney fibrosis and glomerulosclerosis induced by ADR. Mechanistically, ADR induces a robust accumulation of β‐catenin in the nucleus and stimulates its downstream target gene expression. The application of salidroside largely abolishes the nuclear translocation of β‐catenin and thus inhibits its activity. Furthermore, the activation of β‐catenin almost completely counteracts the protective roles of salidroside in ADR‐injured podocytes. Taken together, our data indicate that salidroside ameliorates proteinuria, renal fibrosis and podocyte injury in ADR nephropathy, which may rely on inhibition of β‐catenin signalling pathway.Salidroside is a major phenylethanoid glycoside in  As for the kidney, Wnt/β‐catenin regulates nephron formation during embryogenesis and involves in injury repair and pathogenesis of kidney diseases.In the present study, the animal model of ADR nephropathy, a widely recognized rodent model for proteinuric kidney diseases, was employed to examine the therapeutic mechanisms of salidroside. The current data showed that salidroside improves kidney structure and function in ADR nephropathy, and these beneficial roles are likely dependent upon the inhibition of β‐catenin signalling pathway.22.1Salidroside  was from National Institute for Food and Drug Control . ADR  and the antibody anti‐α‐tubulin  were provided from Sigma‐Aldrich . The antibodies anti‐nephrin , anti‐podocin , anti‐β‐catenin , anti‐fibronectin , anti‐collagenⅠ and anti‐β‐Actin  were purchased from Abcam . The antibodies anti‐α‐SMA (smooth muscle actin) , anti‐Lamin A/C , anti‐MMP7  , anti‐AGT (angiotensinogen) , anti‐PAI‐1 (plasminogen activator inhibitor‐1) , anti‐Axin2 , anti‐Cyclin D1  and anti‐Snail  were from Cell Signaling Technology . Lipofectamine 2000  was from Invitrogen . Recombinant IFN‐γ (interferon γ)  was obtained from ThemoFisher Scientific .2.2The conditionally immortalized mouse podocyte cell line was kindly provided by Prof. Chuan‐Ming Hao . The cell culture medium was composed of RPMI‐1640 medium, 10% fetal bovine serum (FBS) and recombinant IFN‐γ. Cells were cultured at 33°C in cell incubator. To induce differentiation, recombinant IFN‐γ was removed from culture medium and podocytes were grown at 37°C.2.3−1 d−1 salidroside. Urine and kidney tissues were collected at 5 weeks for further analysis.All experiments involving animals were approved by the Institutional Animal Care and Use Committees of Nantong University . The mouse model of podocyte injury and proteinuria was accomplished by injection of ADR via tail vein at the dosage of 10 mg/kg body wt.2.4Urine creatinine, blood urea nitrogen and albumin were analysed by mouse ELISA (enzyme linked immunosorbent assay) kits (Sigma‐Aldrich) according to the manufacturer's instructions.2.5Kidney samples were fixed in 4% paraformaldehyde at 4°C for 24 hours. The paraffin‐embedded kidney tissues were cut into 3 μm thickness and stained with Masson‐trichrome and Periodic acid‐Schiff (PAS) staining for histological analysis. Quantitation of the fibrotic area was carried out by computer‐aided morphometric analysis software (Image‐pro plus 6).2.6’‐diaminobenzidine tetrahydrochloride substrate. For immunofluorescence, slides were blocked with 10% bovine serum for 30 minutes and immunostained with the primary antibodies.Heat‐mediated antigen retrieval was performed using citrate buffer. Slides were then incubated with the primary antibodies (anti‐nephrin and anti‐podocin) overnight at 4°C. Finally, the colour reaction was initiated by adding 3, 32.7The preparation of kidney samples for transmission electron microscope was described elsewhere.2.84P2O7; 100 mmol/L NaF; 10 mmol/L Na3VO4; 10 mmol/L EDTA; 10 mmol/L EGTA; 10 μg/mL Leupeptin; 1% NP‐40; 10 μg/mL Aprotinin; 20 nmol/L Okadaic acid and 2 mmol/L PMSF (phenylmethanesulfonyl fluoride). Then lysates were subjected to centrifugation at 13 800 g at 4°C for 20 minutes. The supernatant was collected and protein concentration was analysed by a Protein Assay Kit (Bio‐Rad). Samples were boiled for 5 minutes at 100°C in 1X Laemmli buffer.Tissues or cells were homogenized with a homogenizer  in ice‐cold lysis buffer, which was composed of 25 mmol/L Tris‐HCl, pH 7.4; 50 mmol/L Na2.9g. The supernatant was discarded and leaving pellet as dry as possible. Tissue homogenization was performed using tissue grinders in the 400 μL of CER I and homogenate was subjected to vortex for 15 seconds to fully suspend the pellet and placed on ice for 10 minutes, then added cold 22 μL CER II to the tubes. Samples were subjected to vortex for 5 seconds, then incubated on ice for 1 minute and centrifuged at 16 000 g for 5 minutes. The supernatant was assigned as cytoplasmic protein fraction and immediately transferred to a clean ice‐cooled tube. Suspend the insoluble (pellet) fraction in cold 200 μL NER and thoroughly vortex 15 seconds. The vortex was repeated three more times and the mixture was subjected to centrifugation. The resulting supernatant was considered as nuclear protein fraction.The cytoplasmic and nuclear protein extraction was prepared by a commercial kit  according to the manufacturer's instruction. In brief, tissues or cells were washed twice with cold PBS and transferred into Eppendorf tubes for centrifugation at 4°C for 5 minutes at 500 2.10’‐GAGGCACCACTGAACCCTAA‐3’ (forward) and 5’‐CATCTCCAGAGTCCAGCACA‐3’ (reverse); collagen Ⅰ, 5’‐GTAACTTCGTGCCTAGCAACA‐3’ (forward) and 5’‐CCTTTGTCAGAATACTGAGCAGC‐3’ (reverse); Axin2, 5’‐TGACTCTCCTTCCAGATCCCA‐3’ (forward) and 5’‐TGCCCACACTAGGCTGACA‐3’ (reverse); Cyclin D1, 5’‐GCGTACCCTGACACCAATCTC‐3’ (forward) and 5’‐CTCCTCTTCGCACTTCTGCTC‐3’ (reverse).The procedures for RNA extraction and quantitative real time PCR were described previously.2.11Western blot analysis was described previously.2.12post hoc test with SPSS 18.0 software. A value of P < 0.05 was the threshold used for significance.Data are expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using one‐way analysis of variance followed by the Dunnett's 33.1Adriamycin (ADR)‐induced nephropathy is a widely used and well characterized model of initial podocyte injury, albuminuria, hereafter renal inflammation and fibrosis in mice.3.2Next, we examined whether salidroside ameliorates renal injury in ADR‐treated mice. According to PAS staining, ADR treatment induced a significant increase in glomerular extracellular matrix (ECM) deposition, which was reduced by salidroside Figure A,B. Mass3.3ADR treatment has been shown to induce podocyte injury by reducing podocin and nephrin expressions, which are slit diaphragm (SD)‐associated proteins.3.4Previous reports have shown that ADR nephropathy is often associated with Wnt signalling pathway activation.3.5To confirm whether the observed protective effects of salidroside against ADR nephropathy are dependent upon β‐catenin inactivation, we activated β‐catenin pathway and examined the effects of salidroside in ADR‐injured podocytes. Wnt1 is an upstream inducer of β‐catenin signalling pathway To further confirm the above findings, we activated β‐catenin by the transfection of β‐catenin S33Y. As well as Wnt1, β‐catenin S33Y stimulated Axin2 and Cyclin D1 expressions in cultured podocytes Figure A‐C. The 4In this study, we have shown that salidroside attenuated ADR nephropathy in mice. For example, ADR‐induced proteinuria and renal fibrosis were reduced by the application of salidroside; meanwhile, the decreases in nephrin and podocin expressions were counteracted by salidroside. Our data suggest that salidroside might be used as a drug or pro‐drug for preventing proteinuric renal diseases.Chronic kidney disorder (CKD) is a public health problem worldwide, because of the high burden, such as decreased productivity, related adverse outcomes and associated costs.−1 d−1 vs 100 mg/kd/day), the species (rat vs mouse), or the target tissues (the substantia nigra vs the kidney).Mounting evidence indicates that podocyte dysfunction plays a key role for proteinuria. Podocyte injury is manifested in a variety of forms, which include the deletion or mutation of the slit diaphragm‐associated proteins .Recently, Renin‐Angiotensin system (RAS) was characterized as a substrate gene of Wnt/β‐catenin in ADR nephropathy.In conclusion, our data indicate that salidroside mitigates ADR induced podocyte dysfunction, reduces proteinuria and kidney injury; and these beneficial roles may rely on inhibition of Wnt/β‐catenin signalling pathway. These novel findings may provide new insights for developing therapeutic modalities based on salidroside for preventing proteinuric kidney diseases.The authors declare that they have no conflict of interest.


Two weeks after transfection of adeno-associated virus, the rat AF model was established by tail vein injection of acetylcholine- (ACh-) CaCl2 for 7 days, and 1 ml/kg was injected daily. The incidence and duration of AF were recorded with an electrocardiogram. Cardiac function was monitored by cardiac ultrasound. Serum cardiac enzyme was detected by ELISA. The expression of atrial miR-27b-3 and Wnt3a was assayed by quantitative RT-PCR. Atrial fibrosis was determined by Masson's trichrome staining. Expression of atrial Collagen-I and Collagen-III was tested by the immunohistochemical method. Expression of CX43 was measured by immunofluorescence. The expression of Collagen-I, a-SMA, Collagen-III, TGF-β1, CX43, Wnt3a, β-Catenin, and p-β-Catenin was assayed by western blot. Our results showed that miR-27b-3p overexpression could reduce the incidence and duration of AF, alleviate atrial fibrosis, increase atrial CX43 expression, and decrease the expression of Collagen-I, a-SMA, Collagen-III, TGF-β1, Wnt3a, and p-β-Catenin. In addition, the results of luciferase activity assay showed that Wnt3a is a validated miR-27b-3p target in HEK 293T cells. Our results provide a new evidence that miR-27b-3p regulates the signaling pathway of Wnt/β-Catenin by targeting Wnt3a, which may play an important role in the development of atrial fibrosis and AF.MicroRNAs (miRNAs) are regarded as a potential method for the treatment of atrial fibrillation (AF) although its molecular mechanism remains unknown. We found in our previous study that the level of peripheral blood miR-27b-3p and the expression of atrial tissue CX43 were both significantly downregulated in AF patients. In the present study, we propose and test this hypothesis that overexpression of miR-27b-3p attenuates atrial fibrosis, increases CX43 expression, and regulates the signaling pathway of Wnt/ Atrial fibrillation (AF) is a clinically frequently seen cardiac arrhythmia, and its incidence increases with aging and accompanies heart diseases . In receAtrial structural remodeling and electrical remodeling are two important features of AF, which can lead to the occurrence and maintenance of AF. In turn, AF can aggravate atrial structural remodeling and electrical remodeling . Recent β-Catenin is a conserved cellular signaling system in animals that regulates cell survival, death, and proliferation and plays an important role in many human diseases  2014-0003). The experiments were subject to approval by the Ethics Committee of Experimental Animals, Guangxi Medical University.Sprague-Dawley male rats, weighing 200-250 g and 8 weeks old, were raised under standard experimental conditions, 20°C-25°C, 50%-60% RH, and 12 L/12 D photoperiod. The experimental rats were supplied clean drinking water and food 2 (60 μg/ml ACh and 10 mg/ml CaCl2) daily via the tail vein at 1 ml/kg for 7 days; (3) AAV-NC group: rats in this group were injected with AAV-NC (2 × 1011 vector genome (vg) particles/per rat) via the tail vein. 14 days after injection, these rats were injected with acetylcholine- (Ach-) CaCl2 (60 μg/ml ACh and 10 mg/ml CaCl2) daily via the tail vein at 1 ml/kg for 7 days; (4) AAV-miR-27b-3p group: rats in this group were injected with AAV+miR-27b-3p (2 × 1011 vector genome (vg) particles/per rat). 14 days after injection, these rats were injected with acetylcholine- (ACh-) CaCl2 (60 μg/ml ACh and 10 mg/ml CaCl2) daily via the tail vein at 1 ml/kg for 7 days; and (5) AAV-miR-27b-3p-antago group: rats in this group were injected with AAV-miR-27b-3p-antago (2 × 1011 vector genome (vg) particles/per rat) via the tail vein. 14 days after injection, these rats were injected acetylcholine- (ACh-) CaCl2 (60 μg/ml ACh and 10 mg/ml CaCl2) daily via the tail vein at 1 ml/kg for 7 days.40 rats were divided into five groups randomly: (1) sham group: rats in this group were injected with 0.9% of normal saline daily via the tail vein at 1 ml/kg for 7 days; (2) AF group : rats inBefore tests, rats were paralyzed with pentobarbital sodium (40 mg/kg). The limb leads of a 12-lead electrocardiogram were inserted into the animal's four limbs and the nonsinus rhythm was recorded and excluded. Seven days after model construction, the rats were subjected to electrocardiogram test again, and the incidence and duration of AF were recorded.β-actin were designed and synthesized by TaKaRa   and miRcute miRNA isolation kit  following the manufacturer's instruction, respectively. Quantitative detection of total RNA and miRNA was performed by using a NanoDrop 2000 spectrophotometer . Further detection of miR-27b-3p and Wnt3a levels and RNA reverse transcription to cDNA using miRcute Plus miRNA First-Strand cDNA Synthesis Kit  and RevertAid First Strand cDNA Synthesis Kit  were performed according to their respective protocols. The primers of miR-27b-3p, Wnt3a, U6, and , Japan) . The RT-http://www.targetscan.org/) to predict and analyze the binding sites of miR-27b-3p and Wnt3a as shown in Figures 4 cells/well. The cells were incubated at 37°C and 5% of CO2 in a cultivation chamber. miR-27b-3p mimics or miR-27b inhibitor was transfected for 48 h according to the user's manual of Lipofectamine®3000 Transfection Reagent. The luciferase activity was tested with the dual-luciferase reporter system .We used TargetScan 7.2 , left ventricular fractional shortening (FS), left ventricular end-systolic diameter (LVESD), and left ventricular end-diastolic diameter (LVEDd) of these animals were analyzed with the MS400 biological signal analysis system . The mean value of three cardiac cycles was used for statistical analysis.Arterial blood was collected at the apex with heparin anticoagulation tube and then centrifugated by 8000g at 4°C for 10 min. Supernatant was collected to determine the levels of cTnT and CK-MB with ELISA reagent kit according to manufacturer's instruction.The hearts of the rats were quickly removed under anesthesia and the blood was washed with PBS. The left atrial tissue was isolated and fixed with 4% of paraformaldehyde. The sample was imbedded into paraffin and cut into 3 mm thick slices. The slices were stained with Masson's trichrome staining and observed under an optical microscope . The samples were observed at × 400, and three visual fields were tested in each sample. Results were analyzed with Image-Pro 6.0 software.3 mm thick sections were incubated overnight at 4°C using Collagen-I and Collagen-III primary antibody (dilution to 1 : 150). After incubation with the primary antibodies, the sections were washed 3 times at room temperature for 5 min each time and it was incubated with a secondary antibody for 2 h at room temperature. The samples were observed under the optical microscope  at ×400, and three visual fields were tested randomly in each sample. Results were analyzed with Image-Pro 6.0 software.To determine the expression site of CX43, every group was subjected to immunofluorescence test. Sections 3 mm thick were taken and blocked with 10% serum for 1 h. Colocalization detection was then performed by incubating overnight at 4°C with CX43 primary antibody (diluted to 1 : 150). After the incubation of the primary antibody, the sections were washed 3 times at room temperature for 5 min each time. Then, it was incubated with a secondary antibody for 2 h at room temperature. DAPI staining was performed for 10 min before the samples were observed using an optical microscope , at ×400 and three visual fields were tested randomly in each sample. Results were analyzed with Image-Pro 6.0 software.β1, CX43, Wnt3a, β-Catenin, p-β-Catenin, GAPDH, and β-Tubulin for 6 h. Concentration of the primary antibodies were 1 : 2000 for Collagen-I, Collagen-III, TGF-β1 and β-Catenin; 1 : 500 for p-β-Catenin; and 1: 1000 for a-SMA, CX43, Wnt3a, GAPDH and β-Tubulin. Concentration of the secondary antibody was 1 : 14000 and it was incubated for 2 h at room temperature. The signal was tested with the chemiluminescence system (Amersham Pharmacia).Equal amount of left atrial tissue was collected and lysed in the RIPA buffer of phenylmethylsulfonylfluoride (PMSF). Protein content was quantified with the BCA reagent kit and then subjected to SDS-PAGE. The protein was subsequently transferred to the PVDF membrane. The PVDF was incubated by Collagen-I, a-SMA, Collagen-III, TGF-P < 0.05 was considered as statistical significance.Data are expressed as mean ± standard deviation and data were analyzed using Student's t-test or one-way ANOVA. Statistical analysis was performed using Prism 6.0 software package. As shown in The results of cardiac ultrasound showed no significant changes in the cardiac function in the AF group, AAV-NC group, AAV-miR-27b-3p group, and AAV-miR-27b-3p-antago group compared to the sham group as shown in Figures As shown in Figures Masson's trichrome staining test showed that the atrial fibrosis level of the AF group increased significantly compared with the sham group, while it was reduced significantly in the AAV+miR-27b-3p group compared with the AF group. However, a higher atrial fibrosis level was observed in the AAV-miR-27b-3p-antago group compared with the AF group as shown in Figures 5(b)Results of immunohistochemical test showed that the expression level of Collagen-I and Collagen-III in the AF group increased significantly compared with the sham group, in which their expression levels decreased significantly in the AAV-miR-27b-3p group compared with the AF group. However, the expression level of Collagen-I and Collagen-III in the AAV-miR-27b-3p-antago group increased significantly compared with the AF group Figures β1 in the AF group increased significantly compared with the sham group, while their expression decreased significantly in the AAV-miR-27b-3p group compared with the AF group. However, the expression of Collagen-I and TGF-β1 in the AAV-miR-27b-3p-antago group increased significantly compared with the AF group  to predict and analyze if Wnt3a was the target gene of miR-27b-3p As shown in 2+ and K+, which further affects the remodeling and structural change of GJPs. Such changes may promote the maintenance of AF. In recent years, some miRNAs such as miR-25, miR-106, miR-223, miR-133, and miR-328 have been proven to be related to the electrical remodeling [The pathogenesis of AF is rather complicated, and current research suggests that it mainly involves structural remodeling, electrical remodeling, metabolic abnormalities, and neurohormone and molecular changes , 21. In 2-induced AF rat model. Our results showed that miR-27b-3p expression was downregulated in the left atrium of the AF rat model, while the markers of fibrosis Collagen-I, a-SMA, and Collagen-III were upregulated, and the degree of atrial fibrosis increased significantly. In contrast, miR-27b-3p overexpression could alleviate atrial fibrosis and reduce the susceptibility and duration of AF. However, there was no significant change in the cardiac function and myocardial injury markers (cTnT and CK-MB). Based on the pathophysiology perspective, we hypothesize that the animal's cardiac function is still in the compensatory period, further speculating that this is related to the duration of AF and the stage of development of AF. In the next stage, we will extend the duration of AF and further observe the changes in the cardiac function and myocardial injury markers in experimental animals.In the study, we constructed successfully the acetylcholine- (ACh-) CaClβ-Catenin is a key factor in the signal transduction, mediating the canonical Wnt/β-Catenin pathway. It regulates the transcription of fibrosis-related genes in the Wnt pathway such as fibronectin, matrix metalloproteinases-7 (MMP-7), plasminogen activator inhibitor-1 (PAI-1), twist, and snail [β-Catenin and TGF-β signaling pathway regulates the fibrosis process in a cross-talk pattern [β-Catenin regulates the fibrotic diseases. It was found in recent years that there is a cross-talk function between the signaling pathway of Wnt/β-Catenin and CX43 [β-Catenin pathway. Overexpression of CX43 can reduce the transcriptional activity of β-Catenin protein in lithium-stimulated neonatal rat cardiomyocytes [β-Catenin protein and increased the expression of target genes of the signaling pathway of Wnt/β-Catenin [β-Catenin pathway and subjected to its positive regulation and translation [β-Catenin can upregulate CX43 expression, and activation of Wnt/β-Catenin signaling pathway can upregulate CX43 expression in mouse embryonic stem cells and further induce cardiac differentiation [β-Catenin could block mesenchymal stem cell- (MSC-) induced upregulation of CX43 and improve cardiac transduction, indicating the MCSs could alleviate the incidence of arrhythmia by activating the canonical signaling pathway of Wnt/β-Catenin [Wnt protein family is a secreted lipid-modified glycoprotein that can regulate a lot of intracellular signaling transduction cascade. nd snail , 30. In  pattern . Therefoand CX43 ; as an umyocytes . In addi-Catenin . It was nslation . In cardntiation . In addi-Catenin .β-Catenin, and TGF-β1 expression in the left atrium were significantly increased, and CX43 expression was significantly decreased. Overexpression of miR-27b-3p reduced Wnt3a, p-β-Catenin, and TGF-β1 expression levels and increased CX43 expression. We therefore speculate that there may be a regulatory effect between miR-27b-3p and the signaling pathway of Wnt/β-Catenin and CX43. No matter if CX43 negatively regulates the canonical signaling pathway of Wnt/β-Catenin as an upstream regulatory factor, or as a downstream target gene of the canonical signaling pathway of Wnt/β-Catenin, and is subjected to its positive or negative regulation in term of transcription and translation, it was obvious condition-dependent according to literature reports. Therefore, we speculated that the regulatory relationship between the signaling pathway of Wnt/β-Catenin and CX43 depends on disease specificity and tissue specificity, as well as different stages of disease progression.In the present study, we found that Wnt3a, p-β-Catenin. For instance, miR-708-5p-, miR-217-5p-, and miR-499-5p-mediated regulation of the Wnt/β-Catenin signaling pathway is probably a potential pathophysiological mechanism of arrhythmogenic cardiomyopathy (AC) [β-Catenin [β-Catenin by targeting Wnt10a [β-Catenin [β-Catenin signaling pathway [β-catenin pathway and plays an important role in the differentiation of laryngeal carcinoma by targeting GSK-3β [β-catenin to accumulate in the nucleus by targeting APC and activate Wnt/β-catenin pathway to promote odontoblastic differentiation [In recent years, studies have found that miRNAs participate in the process of various fibrotic diseases by regulating the signaling pathway of Wnt/thy (AC) ; miR-375-Catenin . In anotg Wnt10a ; miR-29a-Catenin . Further pathway –43. miR-g GSK-3β ; miR-27 ntiation .β-Catenin. We therefore believed that miR-27b-3p could negatively regulate the expression of Wnt3a, further affecting the activity of the Wnt/β-Catenin signaling pathway. It is further speculated that miR-27b-3p overexpression probably has alleviated the atrial fibrosis of AF rats through the signaling pathway of Wnt/β-Catenin. Furthermore, our results indicated that, as summarized schematically in β-Catenin signaling pathway through targeting Wnt3a could promote atrial fibrosis and the incidence of AF, which is a new molecular mechanism at the miRNA level for AF.In the present study, to further investigate the mechanism that miR-27b-3p overexpression alleviates atrial fibrosis and reduces the incidence and duration of AF, we proved that Wnt3a is a validated target gene of miR-27b-3p using bioinformatics and the luciferase reporter assays. We found that miR-27b-3p overexpression could reduce the expression of Wnt3a and p-β-Catenin through targeting Wnt3a. Therefore, miR-27b-3p probably supplies a new valuable target for gene therapy of AF.Our results supplied a new evidence that downregulated miR-27b-3p could promote atrial fibrosis and occurrence of AF by regulation of the signaling pathway of Wnt/


Contact-angle measurements were used to evaluate the wetting properties of the epoxy, epoxy/TiO2/epoxy and epoxy/FAS-TiO2/epoxy coatings. The epoxy and epoxy/TiO2/epoxy coatings were hydrophilic compared with the strongly hydrophobic epoxy/FAS-TiO2/epoxy coating. The average surface roughness (aS) of the epoxy/FAS-TiO2/epoxy coating was higher than that of the epoxy/TiO2/epoxy coating due to the formation of agglomerates. The biocompatibility evaluation revealed that the cell attachment was significantly higher on the epoxy/FAS-TiO2/epoxy and epoxy/TiO2/epoxy coatings compared with the pure epoxy coating. We also observed improved antibacterial properties for the epoxy coatings with the addition of both TiO2 and FAS-TiO2 nanoparticles.Epoxy/TiO Improved cell attachment and resistance to bacterial adhesion are two critical characteristics to consider in an effective biomaterial design. The appropriate surface modification of the substrate is therefore crucial for a desirable biological response. Various techniques, such as the application of polymer coatings , electroThe biocompatibility of a material is related to cell behaviour on contact with the surface where the surface characteristics of materials such as surface topography, chemistry or surface energy, play an essential part in the adhesion process. The quality of this first phase of the cell-material interaction influences and enables good proliferation and differentiation of the cells on the surface . This mo2 coatings, which are based on the photocatalytic activity of anatase TiO2 [2 is used in many applications where surfaces cannot be cleaned with conventional methods and have to inhibit the growth of pathogenic bacteria [2 with steady antibacterial activity, even under long-term exposure to UV, such coatings are highly applicable for self-cleaning applications [2 is the most benign with respect to the environment and human health [Furthermore, the effect of surface roughness as a critical factor that directly influences bacterial adhesion has also been widely studied . It has ase TiO2 ,17,18. Dbacteria . Due to 2/epoxy coatings with as-received and fluoroalkylsilane (FAS) functionalised TiO2 nanoparticles. We used TiO2 nanoparticles in the form of anatase, which are suitable for applications in self-cleaning and antibacterial coatings due to their photocatalytic properties [2/epoxy coatings on AISI 316L stainless steel. We correlated the surface properties with the amount of attached cells and/or adhering bacteria. The biocompatibility of the coatings was evaluated by bone osteosarcoma cell attachment and the antibacterial properties by the adhesion of Escherichia coli (E. coli).In our study, the surface topography and wettability were controlled by preparing TiOoperties . We studSubstrate.—Austenitic stainless steel AISI 316L  was used as a substrate. Discs of 25-mm diameter were grinded with SiC emery paper up to 4000 grit, diamond polished up to 1 µm, ultrasonically cleaned with ethanol and dried in warm air.2 nanoparticles and fluoroalkylsilane  functionalised TiO2 nanoparticles in the form of a sandwich structure: Epoxy/TiO2/epoxy coating and epoxy/FAS-TiO2/epoxy coating. In the first step, we applied biocompatible epoxy , which was mixed in the wt% ratio 100:45, spin-coated onto the substrate and cured for 3 h at 65 °C. In the next step, two sizes of TiO2 nanoparticles, 30-nm (Cinkarna Celje) and 100-nm  were applied to achieve a dual-size effect. Three separate deposits of 20 μL of 3 wt% TiO2 nanoparticle ethanol solution (30 nm and 100 nm) were alternately spin-coated on a primary epoxy layer. The coating was finalised with the top epoxy layer. The same procedure was repeated with the functionalized TiO2 nanoparticles, which were prepared in 1 vol% fluoroalkylsilane  ethanol solution. The reference coating was pure epoxy coated on the AISI 316L substrate.Coating preparation.—Two sets of coatings were prepared on the AISI 316L substrate with as-received TiOaS:xL and yL are the acquisition lengths of the surface in the x and y directions and z is the height. The size of the analysed area was 714 × 542 μm2.Surface roughness.—An optical 3D metrology system, Alicona Infinite Focus (Alicona Imaging GmbH), was employed for the surface-roughness analysis. Three measurements per sample were performed at a magnification of 20× with a lateral resolution of 0.9 μm and a vertical resolution of about 50 nm. The IF-MeasureSuite (Version 5.1) software was used to evaluate the average surface roughness, 2/epoxy- and epoxy/FAS-TiO2/epoxy-coated AISI 316L substrate were performed using a surface-energy evaluation system . The volume of the water droplets of 5 μL was applied and the average contact angle was determined from five measurements on each surface using a Young-Laplace fitting. The measurements were carried out at 20 °C and ambient humidity.Contact-angle measurements.—The static water contact-angle measurements on the epoxy-, epoxy/TiO® CRL-1427™, Krasteva Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Sofia, Bulgaria)) were used for the assessment of the cytocompatibility/biocompatibility on sterilized epoxy-, epoxy/TiO2/epoxy- and epoxy/FAS-TiO2/epoxy-coated steel substrates. The MG63 cells were cultured under controlled conditions  in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with fetal bovine serum  and 4-mM L-glutamine . The cells were sub-cultured once a week or when they reached 65–70% confluence. Before harvesting with Trypsin-0.25% EDTA  for approximately 10 min at 37 °C, the cells were washed three times with Phosphate Buffered Saline without Ca2+ and Mg2+ . The cells were then resuspended in the growth medium, centrifuged at 200 g for 5 min, and plated at a seeding density of 3 × 104 cells/cm2 in 6-well plates  containing the studied specimens, all in 3 mL of growth medium (27 × 104 cells/well) and incubated for 24 h under controlled laboratory conditions (5% CO2/95% air at 37 °C).Cell adhesion and viability.—Bone osteosarcoma cells  and 2-μg/mL Propidium iodide (stains the nuclei of the non-viable cells red) for 20 min. Fluorescent microscope  was used to observe and count stained cells. Twenty images per sample were randomly taken at 100× magnification. The free software program ImageJ was used for quantitative image analyses of the density of the attached cells and the cell viability. The results were normalized to the sample’s surface area.2PO4 · 2H2O and Na2HPO4·2H2O; all the chemicals from Merck, Germany). The samples were washed in the buffer for 3 × 10 min and post-fixated with 1% osmium tetroxide (OsO4) , followed by washing in the buffer for 3 × 10 min. The samples were dehydrated through an alcohol gradient (30%–100%)  with each step lasting 10 min. Further dehydration steps were conducted with a mixture of Hexamethyldisiloxane  and absolute EtOH , 3:1  and absolute HMDS (10 min) and HMDS (2 h), which was finally left to evaporate.Scanning Electron Microscopy (SEM).—SEM analysis using a FE-SEM JEOL JSM-6500F  was employed to investigate the surface morphology of the coatings as well as the distribution, shape and morphology of the attached M63 cells. Prior to SEM imaging, the specimens were processed for fixation. After 24 h of incubation, the cell-culture medium was removed, the cells were washed three times with PBS and fixed for 24 h at 4 °C using a modified Karnovsky fixative, composed of 2.5% glutaraldehyde  and 0.4% paraformaldehyde  in a 0.1-M Na-phosphate buffer  were randomly taken and the results from the cell adhesion, the viability assay and the biocompatibility were expressed as the arithmetic mean ± standard deviation (SD). The data were tested with the non-parametric Mann-Whitney U test and the calculations were performed with Statgraphics Plus 4.0. All the data were submitted for an analysis of variance (ANOVA) and Duncan’s multiple range test, where appropriate.E. coli (DSM 1576) was grown overnight in TSB (tripton soy bujon), diluted to 0.5 McFarland and 1 mL applied on each tested plate. After 4 h of incubation at 37 °C the plates were washed twice with a sterile physiological solution. To remove the bound bacterial cells the plates were exposed to sonication (60 s); . The obtained sonicate was tenfold diluted and appropriate dilutions inoculated on TSA in parallel. After 24 h of incubation at 37 °C the colony-forming units were counted.Antibacterial evaluation.—The antibacterial activity of the treated material was determined as a decrease in the proportion of attached cells. 2/epoxy and epoxy/FAS-TiO2/epoxy coatings. The main difference between the two coatings was in the length scales of the average size of the nanoparticle agglomerates. An epoxy/FAS-TiO2/epoxy coating is characterised by the formation of agglomerates up to a few tens of microns colγ  [θW measured on the same solid surface, the constant β and the solid surface tension sγ values were determined using the least-squares technique. For the fitting with Equation (2), a literature value of β = 0.0001234 (mJ/m2)−2 was used, as weighted for a variety of solid surfaces. The calculated values for the solid surface energy are listed in For a given value of the surface tension of a probe liquid .8 mN/m)  and θW maS was used to evaluate the morphology difference between the three coatings under investigation. As shown in 2/epoxy surface is covered with large agglomerates, exhibiting high aS = 600 nm. The epoxy/TiO2/epoxy coating, on the other hand, results in a reduced average surface roughness of 320 nm compared with the epoxy/FAS-TiO2/epoxy surface due to more finely dispersed nanoparticles with no visible agglomerates.The average surface-roughness parameter 2/epoxy and epoxy/TiO2/epoxy samples were investigated. The surface characteristics of the materials play an important part in the adhesion process and, consequently, the adhesion phase of the cell–material interaction influences the proliferation and differentiation of the cells on the surface [2/epoxy- and epoxy/TiO2/epoxy-coated, compared with pure epoxy-coated, samples after 24 h of exposure. The epoxy/TiO2/epoxy coating showed a higher degree of cell adhesion compared with the epoxy/FAS-TiO2/epoxy coating (2 nanoparticles was significantly lower (5.9 ± 0.5 cells/mm2 on the epoxy/FAS-TiO2/epoxy and 8.7 ± 3.5 cells/mm2 on the epoxy/TiO2/epoxy) compared with the pure epoxy coating (32.1 ± 7.3 cells/mm2)  .2/epoxy and epoxy/TiO2/epoxy coatings.SEM was used to assess the attachment and morphology of the MG63 cells on all the investigated samples. There were no differences observed in the attachment pattern between the different samples. The cells were randomly attached without any particular direction on the epoxy as well as on the rougher epoxy/FAS-TiOThe cell morphology was analysed in order to understand the cells’ response to the coating. Flattened and well-spread polygonal cells with extended filopodia represent the normal morphology of healthy, well-adhered cells with a high cellular interaction with the substrate compared to the smaller, round-shaped morphology of the non-proliferating/apoptotic-like cells, indicating poor adhesion ,25.2/epoxy (5 ± 2%) and the highest on the epoxy coatings (10 ± 4%). The size of the polygonal cells was mainly 30 ± 8 μm. The round-shaped cells were smaller (10 ± 3 μm) and agglomerated on top of the other flat cells. Mostly, they were not firmly attached to the surface, which indicates that they did not find a proper site or contact to start the adhesion process and proliferation. On rougher epoxy/FAS-TiO2/epoxy and epoxy/TiO2/epoxy coatings, the filamentous protrusions (filopodia) found TiO2 nanoparticles as the anchoring points for the adhesion  and cell interconnections known as tunneling nanotubes (TNTs), not only between the adjacent cells but also among the cells far from each other .A heterogeneous collection of EVs, exosomes and ectosomes, generated by all the cells, differ in size, composition, mechanisms of assembly, regulation of release and play an important role in intercellular communications ,27,28. L2/epoxy and epoxy/TiO2/epoxy coatings promote osteoblast growth. It is expected that the proposed approach to bio-functionalize and induce the antibacterial properties of a stainless-steel surface advance the development of new materials/surfaces for diverse biomedical applications.These findings suggested that the epoxy/FAS-TiOE. coli in a TSB medium for 24 h. The antibacterial properties of the coatings were evaluated by plate counting of the adhered E. coli with and without washing and presented in E. coli colonies decreased with the increased hydrophobicity and surface roughness of the coating. In general, moderate hydrophobic or hydrophilic surfaces are more able to bind bacteria in comparison with superhydrophobic and superhydrophilic surfaces [E. coli adhesion also on the moderately hydrophobic coating (θW = 120°) due to the lowered surface energy by fluorination of the TiO2 nanoparticles. In addition, the superior antibacterial properties of epoxy/ FAS-TiO2/epoxy coating against E. coli compared with the epoxy/TiO2/epoxy and pure epoxy coatings are also governed by an increased surface roughness, which significantly reduced the contact area for bacterial binding and therefore weakens the adhesion. Moreover, the epoxy coatings with the addition of TiO2 and FAS-TiO2 nanoparticles, compared with the pure epoxy coating, resulted in reduced E. coli adhesion due to the known antibacterial effect of anatase TiO2 [2 nanoparticles are attributed to the reactive oxygen species, O2−, OH and HO2 radicals, which are produced by redox reactions between the adsorbed species (such as water and oxygen) and electrons and holes photo-generated by the UV irradiation of the TiO2, even under both natural solar irradiation and ordinary room light [2 surface area, and can be increased by reducing the particle size to the nanoscale [Surfaces with different wettability were exposed to surfaces . Howeverase TiO2 . The antom light . In addianoscale .2/epoxy and epoxy/FAS-TiO2/epoxy, that were applied on the surface of AISI 316L stainless steel. Both coatings are characterised by unique surface properties that differ in terms of wettability and surface roughness. The hydrophilic epoxy/TiO2/epoxy coating prepared with as-received TiO2 nanoparticles forms a smoother surface compared with the hydrophobic epoxy/FAS-TiO2/epoxy coating characterised with FAS-functionalised TiO2 nanoparticles agglomerates. The biocompatibility was evaluated in terms of the cell adhesion, viability and cell morphology of the MG63 cells. We have shown that the cell attachment was significantly higher on the epoxy/FAS-TiO2/epoxy and epoxy/TiO2/epoxy coatings compared with the reference pure epoxy coating. Additionally, the amount of observed dead cells on both coatings with TiO2 nanoparticles was considerably lower compared with the pure epoxy coating. Moreover, the epoxy coatings with the addition of TiO2 and FAS-TiO2 nanoparticles compared with the pure epoxy coating resulted in a reduced E. coli adhesion due to the known antibacterial effect of the anatase TiO2. Finally, we were able to successfully improve the biocompatibility and antibacterial properties of the epoxy coating with the implemented as-received and FAS-TiO2 nanoparticles.We prepared two types of sandwich-structured coatings, epoxy/TiO