H. pylori increased ETS2 transcription factor-mediated Siah1 protein expression in gastric cancer cells (GCCs) MKN45, AGS and Kato III. Siah1 protein level was also noticeably higher in gastric adenocarcinoma biopsy samples as compared to non-cancerous gastric epithelia. Siah1 knockdown significantly decreased invasiveness and migration of H. pylori-infected GCCs. Although, Siah1 could not increase degradation of the cytosolic β-catenin and its nuclear translocation, it enhanced degradation of the membrane-bound β-catenin in the infected GCCs. This loss of membrane-bound pool of β-catenin was not associated with the proteasomal degradation of E-cadherin. Thus, this work delineated the role of Siah1 in increasing invasiveness of H. pylori-infected GCCs.β-catenin has two different cellular functions: intercellular adhesion and transcriptional activity. The E3 ubiquitin ligase Siah1 causes ubiquitin-mediated degradation of the cytosolic β-catenin and therefore, impairs nuclear translocation and oncogenic function of β-catenin. However, the effect of Siah1 on the cell membrane bound β-catenin has not been studied. In this study, we identified that the carcinogenic bacterium  These proteins modulate several cellular functions including angiogenesis, inflammation, cell proliferation, cell migration and apoptosis.11 β-catenin is involved in the cell–cell adhesion, as well as Wnt-signaling in epithelial cells.13 Two separate pools of β-catenin maintain these two very different functions—a nonmembranous cytoplasmic-nuclear pool and a cell membrane pool bound with E-cadherin.14 In the absence of Wnt-signaling, the cytoplasmic β-catenin is degraded by the ubiquitin-proteasomal degradation pathway. This results in the accumulation and translocation of β-catenin to the nucleus and causes transcriptional activation of its target genes.15Siah1 is involved in the p53-mediated degradation of the oncogene β-catenin in the cytosol.17 What happens to the cadherin-bound β-catenin after the complex is dissociated from the adherens junction is not clearly known but it is considered to be either degraded or recycled.18 Another E3 ubiquitin ligase, Ozz-E3, ubiquitinates β-catenin and causes its proteasomal degradation.19 Since there is only one report of a rare inactivating mutation of siah1 in gastric cancer20 and none in any other cancers,21 the cellular function of Siah1 in regulating gastric cancer possibly is not limited to its tumor-suppressive role. This notion is further supported by animal studies, which have shown Siah1 as a tumor-promoter.8Siah1-mediated degradation of cytoplasmic β-catenin is a phosphorylation-independent mechanism. While several studies reported about proteasomal degradation of β-catenin in the cytosolic compartment, very little is known about its proteasomal degradation in the cell membrane adherens junction. However, this is clear that cell membrane-bound β-catenin degradation is associated with the loosening of the cell–cell attachment. The E3 ubiquitin ligase Hakai ubiquitinates cell membrane-bound E-cadherin and β-catenin leading to the internalization of the E-cadherin complex and enhanced epithelial cell migration.H. pylori infection on the expression and activity of Siah1 protein in the infected gastric cancer cells (GCCs). Our findings reveal a novel mechanism of H. pylori pathogenesis wherein H. pylori-mediated upregulation of ETS2 induces siah1 transcription. We observe that Siah1 causes membrane-bound β-catenin degradation and induces invasiveness and migration of infected GCCs.We sought to investigate the effect of H. pylori-infected gastric cancer cells (GCCs), MKN45 cells were infected with a cytotoxin-associated gene pathogenicity island-positive {cag PAI(+)} H. pylori strain 26695 at a multiplicity of infection (MOI) 100 and 200 for 3 h and 6 h. Western blot analysis revealed that although MOI 100 at 6 h post infection (p.i.) and MOI 200 at both 3 h and 6 h significantly induced Siah1 protein, MOI 200 at 6 h was maximally effective  showed that 6 h was the optimal time required for Siah1 protein induction  confirmed that Siah1 messenger RNA (mRNA) was significantly (*P <0.05) enhanced after 2 h p.i. as compared to the uninfected control (cag PAI(+) strain with an isogenic cag PAI(-) mutant strain 8-1 revealed that Siah1 protein induction was a cag PAI-independent event . We identified the presence of an E26 transformation-specific (ETS) binding site (EBS) in the siah1 5′ UTR 22 (To identify the transcription factor responsible for Siah1 upregulation in the 626.1)22 .In vitro binding assay was performed to study ETS2 binding with the EBS of the 5′ UTR of siah1 in uninfected and infected MKN45 cells. WT and EBS-mut oligonucleotides were 5′ biotinylated (H. pylori-infected MKN45 cells were incubated with biotinylated-oligonucleotides coated over magnetic beads. Western blots of bead-bound proteins showed ETS2 binding with the siah1 promoter EBS only in the infected cells (In vivo binding of ETS2 with siah1 EBS was further confirmed by chromatin immunoprecipitation (ChIP) assay. Uninfected and H. pylori-infected MKN45 cells were immunoprecipitated using ETS2 specific antibody. PCR products obtained from the immunocomplex represented the siah1 promoter and 5′ UTR flanking the EBS (S=specific PCR product). PCR product was not obtained from the 5′ far upstream sequence (NS=non-specific PCR product) (inylated . Nucleared cells . Westernproduct) .siah1 transactivation. WT or EBS-Mut siah1 reporter constructs were co-transfected with Renilla luciferase construct phRLTK in MKN45 cells followed by infection with H. pylori for 1 h. Data confirmed that H. pylori significantly increased siah1 transactivation in the WT siah1 EBS-expressing cells as compared to the mut EBS-expressing cells  compared to non-cancer tissues (n=10)  collected from consenting patients. H&E staining and fluorescence microscopy showed marked induction of ETS2 and Siah1 in adenocarcinoma samples (s (n=10) . These dH. felis as this model represents the classical cascade of H. pylori-driven carcinomatous changes observed in humans.25 Eight months p.i. all infected mice showed precancerous lesions represented by marked mucus-gland metaplasia, enhanced ETS2 and Siah1 expression (n=16) as compared to the uninfected tissues (n=16) (C57BL/6 mice were infected with s (n=16) .10 Siah1 enhanced β-catenin degradation in the cytosolic compartment of hepatocytes and thereby reduced its nuclear localization and effectiveness as an oncogenic factor.26 The same study, however, showed that the membrane-bound β-catenin was not affected by Siah1. Siah1 was also reported to degrade cytosolic β-catenin in the cervical epithelial cancer cells.27 To find out the effect of H. pylori infection on β-catenin, we performed western blot of whole cell lysates prepared from H. pylori-infected  or uninfected MKN45 cells. Data indicated that although Siah1 was significantly (*P<0.05) induced by H. pylori, β-catenin level in the whole cell lysate did not change with H. pylori infection (*P< 0.05) downregulated only after 20 h of H. pylori infection  Siah1 protein levels after H. pylori infection at all time points, did not demonstrate any loss of β-catenin in infected cells. P.i., nuclear fractions also showed a time-dependent significant (*P< 0.05) increase in Siah1 but unchanged β-catenin protein level. Confocal microscopy performed on MKN45 cells further confirmed that expression of only the membrane-bound β-catenin was highly reduced 20 h p.i.  blocked H. pylori-mediated membrane β-catenin degradation (ETS2 knockdown on Siah1 protein. siETS2 significantly (*P<0.05) reduced ETS2, as well as Siah1 protein levels in the whole cell lysates (*P< 0.05) decrease H. pylori-driven membrane β-catenin degradation. Data shown in To assess the effect of nfection . With Sinfection . siSiah1nfection . Howeverradation . Next, w lysates . Post si lysates . siETS2 16 and previous studies reported about loss of E-cadherin from the membrane in H. pylori-infected GCCs,29 we wanted to assess E-cadherin status in the membrane of Siah1-overexpressed cells in the presence or absence of H. pylori. For this, MKN45 cells were transfected with the empty vector (pcDNA3.1+) or Siah1 construct. Transfected cells were infected with H. pylori for 20 h in the presence or absence of either vehicle control or 50 μM MG132. Data revealed that neither Siah1 overexpression nor H. pylori promoted membrane-bound E-cadherin loss in GCCs  since MKN45 cells partly grow in clumps.35 pcDNA3.1+ or Siah1 stably-expressing AGS cells were grown in monolayer, a wound was marked and cells were incubated in the presence or absence of H. pylori for various time periods. A significant time-dependent increase in cell migration was observed in Siah1-expressing H. pylori-infected AGS cells than empty-vector-expressing infected cells (H. pylori-induced cell migration (*P< 0.05) cell invasion was observed in siSiah1-expressing infected AGS cells as compared to control duplex-expressing infected AGS cells  H. pylori strain. Our findings corroborate another report by Bebb et al. which shows that the reduction in membrane β-catenin occurs in H. pylori-infected cells without concomitant increase in its cytosolic and nuclear pool.43 However, as only nonphosphorylated CagA interacts with E-cadherin44 while CagA gets phosphorylated in the infected host,46 cell disruption of E-cadherin-β-catenin interaction by CagA in our system is ruled out. We note that loss of membrane-bound β-catenin only takes place in H. pylori-infected GCCs but not in uninfected GCCs. This could be due to increased activity of Siah1 protein in H. pylori-infected GCCs. Research is currently underway in our laboratory to identify the possible role of posttranslational modification(s) in regulating Siah1 activity and whether that is restricted to the cell membrane.This study reveals that Siah1 mediated membrane β-catenin degradation is independent of membrane E-cadherin status. Interestingly, H. pylori-infected GCCs but it does not cause any change in the cytosolic and nuclear pool of β-catenin. These observations are in complete contradiction with earlier findings which have shown that the cytosolic fraction of β-catenin is degraded by Siah1 and thus, Siah1 act as a tumor suppressor.47 As Siah1 can be upregulated in a p53-dependent manner,49 association of Siah1 with apoptosis is also widely accepted. Surprisingly, we find that the level of Siah1 protein in H. pylori-infected GCCs is not dependent on p53 as it is also induced in the p53-null Kato III cells. As basal expression of Siah1 is noticed in uninfected GCCs when ETS2 protein is not expressed, it is clear that p53 and ETS2-independent mechanisms of siah1 transcription exist.Siah1-mediated cytosolic β-catenin degradation is thought to have an overall tumor-suppressive effect. We find that Siah1 protein is induced by ETS2 in 50 Recently we have shown that Siah2 was induced by ETS2 and Twist1 in H. pylori-infected GCCs.51 It is therefore evident from the current study that ETS2 can simultaneously induce both Siah1 and Siah2 proteins in H. pylori-infected GCCs. As Siah1 lacks Twist1-binding site, we speculate that these isotypes can still be differentially regulated since ETS2 and Twist1 expression might vary depending on the staging of gastric cancer. As we have not observed any role of Siah2 in membrane-bound β-catenin degradation in H. pylori-infected GCCs (unpublished data), target molecules of these Siah isotypes might very well be different. We believe that despite having a common transcription factor ETS2, Siah1 and Siah2 have unique roles in the cell and disease processes.E3 ubiquitin ligases are involved in various cellular processes including cancer and inflammation.H. pylori-induced degradation of cell membrane-associated β-catenin, we believe that Siah1 is a crucial factor regulating gastric cancer progression and metastasis. Stage-dependent variation in Siah1-β-catenin interaction might exist that can determine the course of treatment-outcome. For this, further studies with patient samples are required which will surely enrich our current knowledge of the molecular basis of H. pylori-mediated gastric cancer pathogenesis.Since this work uncovers a new mechanism of 53 Full-length wild type (WT) human Siah1 cDNA was subcloned in HindIII/XhoI restriction sites of eukaryotic expression vector pcDNA3.1+ . ETS2 construct (#28128) was procured from Addgene, MA, USA. The full length human siah1 promoter was cloned into the pGL3 basic vector  using restriction sites KpnI and HindIII. The promoter construct siah1 WT was used as a template to generate mutation at the ETS2-binding site (EBS) using Quik Change site-directed mutagenesis kit . Constructs were confirmed by sequencing. Primer sequences are shown in The human adenocarcinoma GCCs MKN45, Kato III, AGS were cultured and maintained, as described earlier.H. pylori strains 26695, 8-1 and H. felis strain 49179  were cultured and maintained as reported previously.53 Unless otherwise specified, GCCs were infected with strain 26695 at 200 MOI for the indicated period. To study proteasome-mediated degradation, cells were co-treated with the proteasome inhibitor MG132 for 6 h at 50 μM dose.H. pylori eradication therapy were excluded. The investigation was carried out in compliance with the Helsinki Declaration (2013) of the World Medical Association.Endoscopic biopsy samples  were collected from consenting patients who were suffering from stage III gastric adenocarcinoma following a National Institute of Science Education and Reseach (NISER) Review Board-approved protocol. Pathologically-confirmed gastric cancer cases with paired non-cancerous gastric tissues were included for the study. Patients with a history of previous 6 MKN45 cells were seeded in 6-well plates. On the next day, cells were transfected with 2.5 μg of plasmid DNA, 5 μl of P3000 reagent and 7.5 μl of Lipofectamine3000 reagent (Invitrogen). After 36 h, cells were infected with H. pylori. To knockdown expression of ETS2 and siah1, 0.2 × 106 MKN45 cells were seeded in 6-well plates. On the next day, cells were transfected with 50 nM siRNA of ETS2 or Siah1 along with 10 μl of Lipofectamine3000 reagent (Invitrogen). To generate stable cell lines, MKN45 or AGS cells were seeded in 96 well plates 18–24 h before transfection. Stable transfectants were established with G418 selection.To transiently express Siah1, 1 × 1051Total RNA was isolated from MKN45 cells after 30 min and 2 h of infection using an RNeasy kit . cDNA was synthesized and real-time RT-PCR was performed as described previously.siah1 EBS oligonucleotide (WT or Mut) coated on streptavidin-coated superparamagnetic beads . Binding assays were performed as described previously.51 Oligos are shown in Nuclear lysates were incubated with 5′ biotinylated double-stranded (ds) 51 Chromatins were immunoprecipitated using ETS2 antibody . PCR amplification was performed to analyze the in vivo binding of ETS2 to siah1 5′ UTR EBS. Primers used for ChIP assay are shown in ChIP assay was performed using QuikChIP chromatin immunoprecipitation kit  according to the manufacturer’s protocol.siah1 promoter after H. pylori infection. Cells were co-transfected either with the WT or ETS2-Mut siah1 luciferase constructs (cloned in pGL3 basic vector) and the phRLTK Renilla luciferase constructs at a ratio of 50:1 using Lipofectamine 2000 reagent (Invitrogen). For another set of experiment, cells were co-transfected with WT siah1 luciferase construct along with ETS2 overexpression plasmid and phRLTK Renilla luciferase construct at a ratio of 25:25:1 using Lipofectamine 2000 reagent (Invitrogen). At 36 h of transfection, uninfected or 2 h H. pylori-infected cells were lysed with passive lysis buffer (Promega) and analyzed for luciferase activity as described earlier.51Dual luciferase assays was performed to study activity of H. pylori-infected MKN45 cells using NE-PER Nuclear and Cytoplasmic Extraction Reagents . Around 2 × 106 MKN45 cells were seeded in 60 mm cell culture dish 24 h before infection. Postinfection, cytoplasmic and nuclear fractions were isolated as per manufacturer’s instruction, whereas membrane proteins were isolated from the cytoplasmic extract after further centrifugation at 16000g for 45 min at 4 °C. Whole cell extracts were prepared from uninfected or H. pylori-infected GCCs. For immunoblotting, proteins were resolved on SDS-PAGE gel and blotted onto PVDF membranes. The following primary antibodies were used: Siah1 (1:250) , ETS2 (1:1000) , E-cadherin (1:1000)  and β-catenin (1:5000) . α-tubulin (1:5000) , histone-deacetylase 1 (HDAC1) (1:1000) , Pan-cadherin (1:1000)  antibodies were used for normalization of protein loading. Immunoreactive bands were detected as described earlier.51Nuclear, cytoplasmic and membrane proteins were isolated from 6 MKN45 cells were seeded in 100 mm cell culture dish 24 h before infection. Membrane fractions were isolated and were incubated with Siah1 primary antibody (Novus) for overnight at 4 °C. The protein-antibody complex was pulled down using 50% A/G agarose followed by washing with ice-cold phosphate-buffered saline (PBS). Proteins were denatured by using Laemmli buffer  and analyzed by western blotting.For, coimmunoprecipitation assays, 5 × 1051AGS cells were transfected with or control duplex RNA and siRNA of Siah1. Cell migration and invasion assay was performed following a previously-described protocol.siah1 or pcDNA3.1+ were seeded and wound-healing assay was performed as described previously.51To study the effect of Siah1 on the wound-healing property of GCCs, adherent AGS cells were preferred than semi-adherent MKN45 cells. AGS cells stably-expressing +) or ETS2 or Siah1-expressing MKN45 stable cells following a previously-described method.51Anchorage-independent growth was studied by performing soft-agar assay using the empty vector  were either stained with H&E or processed for immunofluorescence microscopy as mentioned below.Four to five week-old both male and female C57BL/6 mice were procured from the National Centre for Laboratory Animal Sciences of the National Institute of Nutrition . The study was performed after getting the institutional animal ethics committee (IAEC) approval provided by NISER . Male and female mice of 7–8 weeks of age were randomly and blindly divided into two groups- uninfected and infected (16 mice in each group) and infected with H. pylori for 20 h or left uninfected followed by fixation and staining.51 Cells were incubated with Siah1  or β-catenin  or E-cadherin  primary antibodies for overnight and Images were taken using a confocal microscope.51MKN45 cells were seeded on glass coverslips 24 h before infection to study endogenous levels of various proteins. For transfection-based assays, MKN45 cells were seeded on coverslips 24 h before transfection and were transfected using Lipofectamine3000 (Invitrogen). Nontransfected and transfected cells were either infected with 51 Fixed sections were incubated with primary antibodies against Siah1  or ETS2  followed by incubation with secondary antibodies. Images were taken following a previously-described method.51 H&E staining was done following standard procedure.For immunostaining, 5 μm thick gastric adenocarcinoma biopsy specimen were fixed as before.In vitro experiments had a minimum of three independent experiments, each with three technical replicates. No statistical method was used to predetermine the sample size. Experiments were performed and analyzed in non-randomized and non-blinded fashion . Statistical analysis of data was performed by Student’s t-test for comparisons involving two groups. Values were given as mean±s.e.m. Statistical significance was determined at *P<0.05. Two-way ANOVA was performed to compare various transfection groups. Tukey’s test was done for post hoc comparisons. There was no estimate of variation within each group.


Also, mice peritoneal epithelial cells (mPECs) were cultured in 4.25% glucose (HG) or combined with 10 μm ICG‐001 (HG+ICG) for 48 h. We found greater thickness of the parietal peritoneum in the PDF‐treated mice. Additionally, lower expression of E‐cadherin, higher expression of Vimentin, β‐catenin, and Snail, and activation of β‐catenin was observed in the mice and in HG‐treated mPECs, all of which were reversed by ICG‐001. The changes in E‐cadherin and Vimentin indicated occurrence of the epithelial‐to‐mesenchymal transition (EMT). Thus, β‐catenin signaling participates in the process of HG‐induced peritoneal fibrosis, and the EMT of peritoneal epithelial cells is one of the underlying mechanisms of this pathological change.Long‐term exposure to peritoneal dialysate with high glucose (HG) leads to peritoneal fibrosis and thus decreases dialysis efficiency. In this study, we explored the role of β‐catenin in this process. C57BL/6 mice received daily intraperitoneal injection with 10% of the body weight of saline (control), 4.25% glucose peritoneal dialysis fluid (PDF), or PDF combined with 5 mg·kg EMTepithelial‐to‐mesenchymal transitionHGhigh glucosemPECmice peritoneal epithelial cellPDFperitoneal dialysis fluidPDperitoneal dialysisPeritoneal dialysis (PD) is an alternative therapy for patients with end‐stage renal disease and is widely used, especially in developing countries. However, long‐term exposure to biologically incompatible PD fluid (PDF) containing high glucose (HG) and its metabolic products tend to interrupt the integrity of the peritoneal membrane, resulting in peritoneal fibrosis and hyperpermeability via the EMT during PD‐related peritoneal fibrosis The epithelial‐to‐mesenchymal transition (EMT) of peritoneal epithelial cells is considered to be one of the mechanisms underlying peritoneal fibrosis, which starts with the disruption of intercellular junctions, loss of apical‐basolateral polarity, and increased migratory, invasive, and fibrogenic features Many factors are closely associated with the EMT in patients undergoing PD, such as uremic toxins and the nonphysiological nature of dialysis fluids In this study, we investigated the role of β‐catenin in PDF‐induced peritoneal fibrosis in C57BL/6 mice and HG‐induced fibrotic change in mice peritoneal epithelial cells (mPECs), and its potential relationship with pathological EMT changes.The parietal peritoneum in the control group was thin and covered with a layer of flattened epithelial cells, while long‐term PDF injection resulted in a significantly thicker peritoneum, a loose subepithelial matrix, and collagen accumulation. 5‐Bromo‐2‐deoxyuridine (BrdU) staining also showed higher cell proliferation at the parietal interface in the PDF group than in the control group Fig. .E‐cadherin is a key epithelial cellular adhesion protein, and Vimentin is an essential component regulating the EMT Immunofluorescence showed that while Vimentin‐positive cells were barely seen in mPECs exposed to normal glucose (NG), they were significantly increased in those incubated in HG Fig. A. WesterAs transcription generators and EMT markers, up‐regulated cytoplasmic β‐catenin and Snail, both of which are products of β‐catenin target genes, translocate into the nucleus l‐glucose, and found no significant difference in β‐catenin level, illustrating that osmotic pressure variations had little effect on the activation of β‐catenin expression .Similar to the experiments on the effect of PDF on the peritoneum, we examined the localization of GSK, β‐catenin, and Snail by immunofluorescence and western blotting analysis of mPECs. We found that both total and phosphorylated GSK‐3β were up‐regulated by HG incubation, while p‐GSK‐3β demonstrated much larger significance Fig. A,B. ParaTo assess whether activation of the β‐catenin pathway contributes to the onset of peritoneal fibrosis, we introduced the specific inhibitor ICG‐001 along with PDF injections. Daily ICG‐001 treatment significantly reduced the thickness of the submesothelial zone, as well as the number of BrdU‐positive cells in the peritoneum  and was carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80‐23). Two‐month‐old male C57BL/6 mice were provided by the Experimental Animal Center in Zhejiang Academy of Medical Sciences . Mice were maintained on standard rodent chow and had free access to food and water under a 12/12 h light/dark cycle, and BrdU was from Sigma . PDF with 4.25% glucose was from Baxter . ICG‐001 was from Tocris Bioscience . Antibodies against β‐catenin, E‐cadherin, and Vimentin were from Cell Signaling Technology . Antibodies against Snail and GSK‐3β were from Abcam .−1·day−1) −1, intraperitoneal) every 2 days. Mice were deeply anesthetized and sacrificed by cervical dislocation after 30 days, and peritoneal tissues were obtained for histopathology.Fifteen C57BL/6 mice were randomly divided into three groups. The mice in the control group were given 10% of their body weight of saline by intraperitoneal injection per day. The PDF mice received 10% of their body weight of 4.25% glucose PDF, while the PDF+ICG mice received the same volume of PDF combined with ICG‐001  for 48 h and then harvested for immunofluorescence staining and protein expression measurement.Mice peritoneal epithelial cells were collected by needle aspiration 5 min after intraperitoneal injection of 2 mL of 0.25% trypsin‐EDTA, and cultured in Dulbecco's modified Eagle's medium (DMEM/F12) with 10% FBS  in a 5% COPeritoneal tissues fixed in 4% paraformaldehyde were dehydrated in ethanol and cleared in xylene, then embedded in paraffin and cut into 4‐μm sections. To evaluate peritoneal fibrosis, sections were stained with Masson's trichrome, and 10 images at high magnification (×400) were assessed for each animal; the average value for five sites was calculated to evaluate the thickness of peritoneal tissue.Antibodies against BrdU, E‐cadherin, and Vimentin were used to label the paraffin sections, and antibodies against β‐catenin and Snail were used to label the frozen sections. Briefly, sections were blocked with 5% goat serum for 1 h, incubated with primary antibodies overnight at 4 °C, and labeled with the appropriate secondary antibodies.imagej .Mice peritoneal epithelial cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X‐100 for 15 min, and blocked in 10% goat serum in PBS for 1 h at room temperature. Antibodies against Vimentin, GSK‐3β, β‐catenin, and Snail were used. Nuclei were counterstained with Hoechst  for 5 min. Images were captured on a fluorescence microscope  and analyzed using Tissues and mPECs were homogenized in RIPA lysis buffer with protease inhibitor cocktail and phosphatase inhibitor cocktail . Supernatants were assayed by western blot. Briefly, proteins were separated on 10% SDS/PAGE and electro‐transferred to nitrocellulose membranes, which were which were then blocked in 5% nonfat milk and probed with primary antibodies against E‐cadherin, Vimentin, β‐catenin, p‐β‐catenin, p‐GSK‐3β, Snail, and β‐tubulin followed by specific secondary antibodies. The membranes were scanned with an infrared imaging system , and the software provided with the system was used to evaluate the band intensity.graphpad prism 6.0, GraphPad Software, Inc., La Jolla, CA, USA). Two‐tailed Student's t‐test was applied (graphpad prism 6.0). P < 0.05 was considered to be statistically significant.Values are mean ± SD, and were compared by two‐way ANOVA with the Bonferroni post‐test from more than two groups (in vitro experiments. HD performed the in vivo experiments and wrote the manuscript. PY and WJ performed the histochemistry experiments and the statistical analysis. RW, LP, JZ, XC, and JZ contributed to the cell culture and histochemistry experiments. Experiments were performed under the supervision of XZ and JS.SJ designed and performed the Fig. S1. The effect of osmotic pressure change on β‐catenin in mPECs.Click here for additional data file.Fig. S2. Lentivirus‐mediated shRNA knockdown of β‐catenin affects the morphology of mPECs.Click here for additional data file.Fig. S3. Lentivirus‐mediated shRNA knockdown of β‐catenin inhibits EMT of mPECs induced by high glucose.Click here for additional data file.


WISP1 was mainly located in cytoplasm of PC12 or SHSY5Y cells. Compared with the negative control group, WISP1 expression reduced obviously in SHSY5Y cells transfected with WISP1 si-RNA. CCK-8 assay showed that pyrroloquinoline quinone (PQQ) had little influence on viability of PC12 and SHSY5Y cells. These results suggested that WISP1 played a protective role after traumatic brain injury in rats, and this effect might be relative to autophagy caused by traumatic brain injury.WISP1, as a member of the CCN4 protein family, has cell protective effects of promoting cell proliferation and inhibiting cell apoptosis. Although some studies have confirmed that WISP1 is concerned with colon cancer and lung cancer, there is little report about the influence of WISP1 in traumatic brain injury. Here, we found that the expression of WISP1 mRNA and protein decreased at 3 d and then increased at 5 d after traumatic brain injury (TBI). Meanwhile, immunofluorescence demonstrated that there was little colocation of WISP1 with GFAP, Iba1, and WISP1 colocalized with NeuN partly. WISP1 colocalized with LC3, but there was little of colocation about WISP1 with cleaved caspase-3. Subsequent study displayed that the expression of  Traumatic brain injury (TBI), also known as brain injury, is mainly caused by external mechanical forces. There were a series of pathological, physiological, and biochemical changes, such as subarachnoid hemorrhage, cerebral blood tube spasms, disturbance of cerebral circulation, and cerebral edema. All these secondary and primary brain injuries led to higher mortality rates. In the present study, Feeney et al.'s  method wWISP1  is a CCN family member, which is more broadly identified with development and tumorigenesis . CCN proWISP1 could express in a variety of tissues, and there is no tissue specialty. Pennica et al. discovered that WISP1 was expressed in many tissues including the adult heart, lung, kidney, small intestine, spleen, pancreas, ovaries, and brain, but there were significance changes about express levels among different tissues . WISP1 cβ-Catenin is a soluble protein located in the cytoplasm which is first found in 1980 by German scientists [β-catenin is homology analogs of armadillo gene of Drosophila in mammals [β-catenin mainly exists in the cell membrane, cytoplasm, or nucleus in complex forms. The location of β-catenin in the cell is related to biological functions. For β-catenin in cell membrane, β-catenin protein in normal mature cells mediated cell adhesion and migration and affected the polarity and integrity of the epithelium [β-Catenin in the cytoplasm passes through into the nuclear, leading to gene transcription, which is closely related to the development and progression of many diseases [β-Catenin in the nucleus promotes transcription of downstream target genes, which accelerate the cell cycle, promote cell proliferation, produce abnormal protein, and eventually lead to the occurrence of tumor [ientists . Subsequ mammals . Intraceithelium . β-Catendiseases . β-Catenof tumor .β-catenin [β-catenin can promote the expression of WISP1 by Wnt1 signaling [β-catenin represent a novel target that has the potential ability to promote tissue proliferation, repair, and regeneration in multiple cell systems [WISP1, as a CCN family member, utilizes protective pathways that include the traditional wingless canonical and noncanonical signaling of Wnt1. In addition, WISP1 can increase the nuclear expression of -catenin . Interesignaling . WISP1 a systems , 19, 20.Pyrroloquinoline quinone (PQQ) is a new oxidoreductase coenzyme which was discovered in the late 1970 of the 20th century. And it is an anionic water soluble compound which exists in almost all biological tissues . PQQ hasβ (GSK-3β), Bim, Bcl-xL, mitochondrial membrane permeability, cytochrome c release, and caspase activation to prevent cell injury [Recent studies have shown that PQQ alters intracellular signaling pathways. For example, when the phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathways are blocked, the protective effects of PQQ will be lost, which implicates that PQQ could regulate neuronal cell survival by (PI3K)/AKT signaling pathway . Similarl injury . PI3K anAlthough many experiments have confirmed that WISP1 has obvious effective on tissue proliferation, repair, and regeneration, there is little report about the effect of WISP1 in traumatic brain injury. In the present study, we try to explore the effect of WISP1 protein on cell expression, which is necessary to treat brain injury by understanding brain injury repair processes. Therefore, we have established a rat model of TBI to try to further research the function of WISP1 in traumatic brain injury.n = 117) were divided into thirteen groups: sham, 1 d, 3 d, 5 d, and 7 d post-TBI and 1 d, 3 d, 5 d, and 7 d post-TBI + 1 mM PQQ or 2 mM PQQ. Referring to Feeney et al.'s [n = 9) were craniotomized only. After all the procedures, animals were returned to their cages and allowed freely to get the food and water. Animals were housed under a 12 h light/dark cycle, and room temperature was kept at 25 ± 0.5°C.Sprague-Dawley (SD) rats (200–250 g) were obtained from the Experimental Animal Center of Nantong University . All animals n = 17 were d18 primers. Primers of WISP1 and GAPDH were designed with primer 5 software and synthesized by biotech company , WISP1 sense primer: 5′-GCCCGAGGTACGCAATAGGAGT-3′ and antisense primer: 5′-CCCACGGTGCCATCAATACAGG-3′; GAPDH sense primer was also designed in same way, GAPDH sense primer: 5′-CAACGGGAAACCCATCACCA-3′ and antisense primer: 5′-ACGCCAGTAGACTCCACGACAT-3′. Quantitative real-time PCR analysis was performed using the LightCycler 96 (Roche), applying real-time SYBR Green PCR technology (Roche). The reaction mixtures contained 5 μl SYBR Master Mix, 0.1 μl of each PCR forward and reverse primer (10 μM), 1 μl cDNA, and 3.9 μl nuclease-free water for a final volume of 10 μl. After one cycle of 95°C for 10 min, 45 PCR cycles were performed, each consists of a denaturation step  and an annealing step . Total RNA concentrations from each sample were normalized by the quantity of GAPDH mRNA. All experiments were repeated at least three times.Total RNA was extracted from the frozen cortex brain tissues with TRIZOL Reagent (Sigma) according to the manufacturer's recommendations. The RNA was reverse-transcribed to cDNA using reverse transcription kit (Thermo) with oligo(dT)β-actin , and β-catenin . The PVDF membrane was washed with TBST (TBS with 0.1% Tween 20) for 10 min at least three times and incubated with corresponding HRP-conjugated secondary antibody for 2 h at room temperature. After washing the PVDF membrane for 10 min at least 3 times, the protein was visualized using Beyo ECL Star .The brain tissues were stored in −80°C; tissue samples were lysed with extraction buffer. Concentration of the protein was tested using bicinchoninic acid assay kit , followed by electrophoresis separation on SDS-PAGE, and then transferred to a PVDF membrane  for 120 min at 100 V. The membranes were blocked with 5% skim milk and then incubated overnight with WISP1 antibody , μm frozen sections were prepared and examined. All sections were blocked with blocking solution  for 1 h at 37°C, incubating overnight with antibody WISP1 , anti-GFAP , anti-NeuN , β-catenin , anti-cleaved caspase-3 , anti-LC3 , and anti-Iba1  and then washing them with 0.01 M PBS for 10 min at 3 times and followed by incubating with a mixture of FITC- or Cy3-conjugated secondary antibodies for 2 h at room temperature and then being washed again in PBS for 10 min at 3 times. The stained sections were examined with a Leica fluorescence microscope .The brain tissue was fixed with 4% paraformaldehyde at 4°C, after the brain tissues sink to the bottom of the bottle and after dehydrating by concentration of gradient , and then, 12 4 cells/well. After being incubated with 50 μM EdU for 24 h, cells were washed with PBS, followed by fixation in 4% formaldehyde for 30 min, then being permeabilizated in 0.5% Triton X-100 for 10 min. After extensive washing with PBS, cells were incubated with Apollo for 30 min and Hoechst for 30 min. Proliferative cells was calculated as the percentage of EdU-positive cells relative to the total number of cells.Cell proliferation was detected by Cell-Light EdU DNA cell proliferation kit  according to the manufacturer's instructions. Astrocytes were cultured into 96-well plates at 4 × 103 cells/well with DMEM in the upper chamber of a 24-well transwell chamber with 8 μm pore size polycarbonate filters (Costar). 10% fibronectin was added in the lower chamber as chemoattractant. Astrocytes migrate for 24 h and then followed by fixation in 4% formaldehyde for 1 h and stained with 0.1% crystal violet. Images were taken with a microscope and five fields were counted.Astrocytes were cultured at 4 × 102.The SHSY5Y and PC12 cells were obtained from the Chinese Academy of Sciences . The cells were cultured in DMEM with 10% FBS at 37°C in an incubator containing 5% COTo silence the expression of WISP1 gene, WISP1 si-RNA was obtained (Invitrogen), WISP1 sense primer: 5′-GGACAUCCAUACACUCAUUTT-3′ and antisense primer: 5′-AAUGAGUGUAUGGAUGUCCTT-3′. si-RNA transfection was performed with lipofectamine 3000 (Invitrogen) according to the manufacturer's guidelines. All data were obtained after being transfected for 72 h.2 at 37°C for 2 h after adding CCK-8 test solution , and the cell viability were detected by using microplate reader at 450 nm.SHSY5Y cells and PC12 cells were seeded onto 96-well plate ahead of time and then treated with different concentrations of PQQ for 24 h. Subsequently, cells were incubated in a humidified atmosphere with 5% COP < 0.05.For each experiment, the mean and standard error were determined. The data were analyzed by means of analysis of variance (one-way ANOVA). Statistical significance was determined at the level of In order to observe the WISP1 mRNA expression after TBI, qRT-PCR was performed. The results showed that, compared with the sham group, WISP1 mRNA slightly decreased at 1 d, reached the minimum at 3 d after TBI, and then recovered at 5 d and 7 d after TBI. Meanwhile, 1 mM or 2 mM PQQ increased WISP1 mRNA expression at 3 d, 5 d, and 7 d post-TBI, but the effect of 1 mM PQQ was obvious to the 2 mM PQQ .To investigate the cell type that WISP1 located, double-labeling immunofluorescence was performed. The colocalizations of WISP1 with astrocyte marker GFAP, neuronal marker NeuN, microglia marker Iba1 were examined respectively. Compared with the sham group, WISP1-positive signals decreased slightly in the control lateral brain and reduced significantly in the ipsilateral brain. However, GFAP positive signals increased slightly in the control lateral brain and had a dramatic increase in the ipsilateral brain. There was a little amount colocalization between WISP1 and GFAP in the sham group. However, obvious colocalizations were observed in the brain at 3 d after TBI including ipsilateral and control lateral brain .NeuN-positive signals obviously decreased in the ipsilateral brain and decreased slightly in the control lateral brain compared with the sham group . Double-At the same time, there was a modicum colocation of WISP1 and Iba1. Compared with the sham group, Iba1-positive signals increased substantially in the ipsilateral brain .Brain injury was often accompanied by the occurrence of apoptosis and autophagy. In the present study, double immunofluorescence was used to detect the colocalization of WISP1 and autophagy marker LC3 and apoptosis marker cleaved caspase-3. Compared with the sham group, LC3-positive signals had a substantial increase in the ipsilateral brain and had no significant change in the control lateral brain after 3 d post-TBI. After treatment with 1 mM PQQ at 3 d post-TBI, WISP1-positive signals rose obviously; LC3 positive signals declined markedly in the ipsilateral brain. Double-labeling immunofluorescence showed that there were a lot of colocalization of WISP1 and LC3 .After 3 d post-TBI, compared with the sham group, cleaved caspase-3 expression was obviously upregulated in the ipsilateral brain and had no significant change in the control lateral brain. After treatment with 1 mM PQQ at 3 d post-TBI, cleaved caspase-3-positive signals declined obviously in the ipsilateral brain. Double-labeling immunofluorescence showed that there was a little colocalization of WISP1 and cleaved caspase-3 .β-catenin protein were performed by Western blot, which indicated that the protein expression trend of WISP1 was similar to that of β-catenin at different time points after TBI. The protein expression of WISP1 and β-catenin decreased slightly at 1 d after TBI, minimized at 3 d post-TBI and rose again at 5 d after TBI, and slightly reduced at 7 d after TBI compared with the sham group  and GSK-3β inhibitor SB21673 (5 μM) with WISP1 (10 ng/ml) were administrated. The experiments showed that the expression of WISP1 reduced with the administration of glutamate and then upregulated after using 50 μM PQQ as well as WISP1 protein. Interestingly, the expression of WISP1 did not increase under the glutamate after blocking the PI3K/GSK-3β signaling pathway, just as the β-catenin did   (50  > 0.05) . At the  > 0.05) .μM, 50 μM, and 100 μM PQQ were added in the cell medium, respectively. EdU assay results showed that PQQ could promote the proliferation of astrocytes inhibited by glutamate   .μM, 50 μM, and 100 μM PQQ could reverse the astrocyte migration inhibited by glutamate  (Transwell migration assay demonstrated that 5  < 0.01) .∗P < 0.05, ∗∗P < 0.01)    is mainly caused by an external mechanical force, which can be divided into primary and secondary damage. TBI can be classified based on the severity or mechanism of injury, as well as other features. In most cases, TBI leads to higher mortality rates and disability; moreover, TBI could cause physical, pathological, and behavioral effects. Therefore, more and more people attracted general attention. Protecting neurons are the main methods to treat cerebral trauma, and the treatment goals are focused in the cell death program targets. However, there is not an effective method to treat TBI so far.WISP1 is a family of secreted proteins and regulate various developmental processes. Wnt1 proteins combine the cell membrane receptor by autocrine or paracrine effects, activate intracellular signaling pathways, regulate target gene expression, and play an important role on cell proliferation, polarity and differentiation, and migration. WISP1 secreted Wnt1-induced protein 1, also known as CCN4, cognating connective tissue growth factor (CTGF) . WISP1 wAstrocyte activation is a common reaction in the central nervous system under many pathophysiological situations, which assume astrocyte cell hypertrophy, swelling, protuberance extension, and glial fibrillary acidic protein expression increase , 27. AstAfter traumatic brain injury, there would be different levels of autophagy. Autophagy has three different categories termed microautophagy, macroautophagy, and chaperone-mediated autophagy . Autophaβ-catenin act as new targets investigated in recent years, which can promote tissues proliferation as well as various cell repair. In regard to the canonical pathways of Wnt1 [β-catenin in neurons that may be mediated through the inhibition of GSK-3β which prevent β-catenin phosphorylation [β activity in other cell systems such as cardiac cells [β-catenin [β-catenin activity and WISP1 regulates its own expression through the ability of WISP1 to control β-catenin phosphorylation and nuclear translocation [β-catenin minimized at 3 d and rose again at 5 d after TBI compared with the sham group. Meanwhile, the treatment with 1 mM or 2 mM PQQ increased the protein expression of WISP1 and β-catenin correspondingly. All these results demonstrated that WISP1 may modulate the protein expression of β-catenin.WISP1 and  of Wnt1 , WISP1 crylation . WISP1 aac cells , 34. In PQQ, also known as methoxatin, has been detected in a wide variety of foods and other sources. PQQ, as an important antioxidant and nutrient, has also begun to focus on nutriology and pharmacology. PQQ belongs to vitamin B . PQQ has


The ST6Gal-I sialyltransferase is upregulated in numerous cancers, and high expression of this enzyme correlates with poor patient prognosis in various malignancies, including ovarian cancer. Through its sialylation of a select cohort of cell surface receptors, ST6Gal-I modulates cell signaling to promote tumor cell survival. The goal of the present study was to investigate the influence of ST6Gal-I on another important receptor that controls cancer cell behavior, EGFR. Additionally, the effect of ST6Gal-I on cancer cells treated with the common EGFR inhibitor, gefitinib, was evaluated.Using the OV4 ovarian cancer cell line, which lacks endogenous ST6Gal-I expression, a kinomics assay revealed that cells with forced overexpression of ST6Gal-I exhibited increased global tyrosine kinase activity, a finding confirmed by immunoblotting whole cell lysates with an anti-phosphotyrosine antibody. Interestingly, the kinomics assay suggested that one of the most highly activated tyrosine kinases in ST6Gal-I-overexpressing OV4 cells was EGFR. Based on these findings, additional analyses were performed to investigate the effect of ST6Gal-I on EGFR activation. To this end, we utilized, in addition to OV4 cells, the SKOV3 ovarian cancer cell line, engineered with both ST6Gal-I overexpression and knockdown, as well as the BxPC3 pancreatic cancer cell line with knockdown of ST6Gal-I. In all three cell lines, we determined that EGFR is a substrate of ST6Gal-I, and that the sialylation status of EGFR directly correlates with ST6Gal-I expression. Cells with differential ST6Gal-I expression were subsequently evaluated for EGFR tyrosine phosphorylation. Cells with high ST6Gal-I expression were found to have elevated levels of basal and EGF-induced EGFR activation. Conversely, knockdown of ST6Gal-I greatly attenuated EGFR activation, both basally and post EGF treatment. Finally, to illustrate the functional importance of ST6Gal-I in regulating EGFR-dependent survival, cells were treated with gefitinib, an EGFR inhibitor widely used for cancer therapy. These studies showed that ST6Gal-I promotes resistance to gefitinib-mediated apoptosis, as measured by caspase activity assays.Results herein indicate that ST6Gal-I promotes EGFR activation and protects against gefitinib-mediated cell death. Establishing the tumor-associated ST6Gal-I sialyltransferase as a regulator of EGFR provides novel insight into the role of glycosylation in growth factor signaling and chemoresistance. N-glycosylated proteins by the ST6Gal-I sialyltransferase [It has long been known that tumor cells display an altered profile of cell surface glycans, however the functional role of glycosylation in regulating tumor cell behavior remains poorly-understood. The changes in tumor glycosylation are not random; instead, a select subset of glycans is consistently enriched in cancer cells. One of these elevated glycan structures is α2-6 linked sialic acid, which is added to nsferase –3. ST6Gansferase –8, and hnsferase –8.One of the central questions regarding ST6Gal-I’s pro-tumorigenic activity is how changes in surface sialylation influence intracellular signaling cascades to modulate tumor cell behavior. We and others have reported that ST6Gal-I regulates the structure and function of a specific cohort of membrane receptors. As examples, ST6Gal-I-mediated sialylation of the β1 integrin drives tumor cell migration and invasion –12, wherIn the present study we identify another important receptor regulated by ST6Gal-I, the receptor tyrosine kinase, EGFR. OV4 ovarian cancer cells with enforced ST6Gal-I expression were subjected to an unbiased kinomics assay, which revealed that EGFR was one of the most differentially activated kinases in cells with upregulated ST6Gal-I. Specifically, EGFR tyrosine kinase activity was markedly enhanced in cells with high ST6Gal-I expression. Based on the kinomics results, we developed several cell model systems with either ST6Gal-I overexpression or knockdown to establish that EGFR is directly α2-6 sialylated by ST6Gal-I. Significantly, we find that ST6Gal-I-mediated sialylation of EGFR stimulates both the basal and EGF-induced activation of EGFR. Furthermore, α2-6 sialylation of EGFR regulates the viability of cells exposed to the EGFR inhibitor, geftinib. These results not only establish a new tumor-promoting function for ST6Gal-I, but also more broadly illuminate the importance of tumor glycans in fundamental tumor cell survival pathways.For routine maintenance of cell lines, cells were grown in DME/F12 (OV4) or RPMI (SKOV3 and BxPC3) media supplemented with 10% fetal bovine serum  and 1% antibiotic/antimycotics (Invitrogen). Cells were transduced with lentivirus encoding either the human ST6Gal-I gene (Genecopoeia) or shRNA against ST6Gal-I . Polyclonal populations of stably-transduced cells were isolated by puromycin selection. Overexpression or knockdown of ST6Gal-I was verified via immunoblot using anti-ST6Gal-I goat polyclonal antibody .www.kinomecore.com) using the PamStation®12 platform  as previously described [www.phosphonet.ca) to identify highly altered kinases that are displayed in bar graph and volcano plots [OV4 EV or OE cells were lysed by adding pre-chilled M-PER Mammalian Protein Extraction Reagent (Thermo-Scientific) containing protease and phosphatase inhibitors (Thermo Scientific). After a 30 min incubation on ice, the lysate was centrifuged at 14,000 rpm at 4 °C, and the supernatant immediately collected and stored at − 80 °C. Global kinase activity (kinomic) profiling was performed in the UAB Kinome Core .Cells were serum deprived for 2 h using media containing 1% FBS prior to treatment with 100 ng/mL rhEGF . Cells were treated for indicated times and lysed using radioimmune precipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors. Total protein concentration was measured by BCA (Pierce). Samples were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were incubated with 5% nonfat dry milk in TBS containing 0.1% Tween-20 (TBST). Immunoblots were probed with antibodies for p-EGFR , or total EGFR , followed by incubation with appropriate HRP-conjugated secondary antibodies . Protein loading was verified using anti-β-tubulin . Protein was detected by enhanced chemiluminescence using the ECL substrate from Pierce (cat# 32106). To visualize basal p-EGFR levels, which are lower than levels of EGF-induced p-EGFR, we optimized blotting conditions by increasing the total amount of protein loaded (> 40 μg) and prolonging film exposure times. We also used a more sensitive ECL reagent for the basal p-EGFR blots . In addition to immunoblotting for p-EGFR and total EGFR, OV4 cells were immunoblotted for total levels of tyrosine phosphorylation using an HRP-conjugated antibody against phospho-tyrosine . Immunoblotting for p-EGFR was performed using three independently-prepared cell lysates, and densitometric quantification of bands from at least three independent blots was achieved using ImageJ software. All bands were normalized to their respective β-tubulin loading controls. Student’s t test was employed to determine significance . Samples were incubated at 4 °C overnight on a rotator. α2-6 sialylated proteins were then precipitated by centrifugation and washed 3 times with ice cold PBS. Precipitates were resolved by SDS-PAGE and immunoblotted for EGFR as described above.Cells were seeded at equal densities into culture plates and allowed to adhere overnight. Prior to gefitinib treatment, cells were serum deprived in 1% FBS-containing media for 2 h and then treated with 1 μM gefitinib  for 24-72 h. Reconstituted Caspase-Glo 3/7 assay reagent  was then added into each well, mixed via orbital shaker, and incubated at room temperature for 45 min. Luminescence was quantified with a BioTek Synergy H1 plate reader. The values represented were normalized to the caspase value for untreated cells. At least two independent experiments were conducted for each cell line.The OV4 ovarian cancer cell line is one of the few cancer lines that lacks detectable ST6Gal-I protein. To evaluate the role of ST6Gal-I in regulating kinase activity, ST6Gal-I was stably overexpressed (OE) in OV4 cells using a lentiviral vector Fig . An emptTo screen for potential changes in cell signaling consequent to forced ST6Gal-I expression, EV and OE cells were subjected to a kinomics assay. As shown in Fig. We next evaluated the activity of specific serine/threonine kinases modulated by ST6Gal-I Fig . In geneGiven that the kinomics assay pointed to enhanced EGFR activation in cells with forced ST6Gal-I expression, we conducted additional analyses of this receptor. We first determined whether EGFR was a direct substrate for ST6Gal-I. To this end, cell lysates were incubated with agarose-conjugated SNA lectin to precipitate α2-6 sialylated proteins. These proteins were resolved by SDS-PAGE and immunoblotted for EGFR. As shown in Fig. To determine whether sialylation-dependent regulation of EGFR was conserved across cancer cell lines, EGFR activity was examined in the SKOV3 ovarian cancer line. ST6Gal-I was either overexpressed (OE) in SKOV3 cells, or stably knocked-down (KD) using a shRNA-bearing lentiviral vector Fig. . SNA preRecent studies suggest that ST6Gal-I has a similar function in multiple cancer types including ovarian, colon, and pancreatic adenocarcinoma. We therefore evaluated EGFR activation in a pancreatic cancer line, BxPC3. ST6Gal-I expression was knocked down (KD) in this line, which led to a loss in α2-6 sialylation of EGFR, as measured by SNA precipitation assay Fig. . ConsistThe combined results in Figs Accumulating evidence suggests that ST6Gal-I is a potent survival factor, providing cancer cells with protection against a variety of microenvironmental assaults. Explicitly, α2-6 sialylation of select receptors inhibits galectin-mediated apoptosis –30, and In the current study, we describe a new survival function for ST6Gal-I in modulating the activity of EGFR. Utilizing an unbiased kinomics assay, we show that overall basal tyrosine kinase activity, a phenomenon well-known to contribute to malignant transformation –36, is pThe role of sialylation in regulating EGFR has been previously investigated. Park et al. manipulated ST6Gal-I expression in colon carcinoma cells, and in concordance with our work, reported that EGFR sialylation prevented geftinib-induced cytotoxicity . HoweverN-glycans on two key Asn residues, Asn-420 and Asn 579, appear to maintain EGFR in a low affinity state. Ablation of Asn-420 glycosylation (via mutagenesis) leads to constitutive EGFR tyrosine phosphorylation and spontaneous oligomer formation [Further studies will be needed to better understand the effect of α2-6 sialylation on EGFR. EGFR has a complex mechanism of regulation involving receptor oligomerization, lipid raft localization, shedding, and dynamic partitioning between cellular compartments including the plasma membrane, endosome and nucleus –45. Manyormation . Eliminaormation . It is tRegardless of the mechanism by which α2-6 sialylation modulates EGFR structure , results herein are consistent with other studies indicating that ST6Gal-I protects against gefitinib-induced cell death . GefitinThe collective data in this report indicate that upregulation of ST6Gal-I, a common feature of cancer cells, promotes heightened activation of EGFR as well as resistance to gefitinib-induced cell death. Considering the immense contribution of EGFR to cancer , 52, 53,


In vitro B16F10 cell experiments presented that STAT3 siRNA PEI complex can enhance cellular uptake and transfection of siRNA, correspondingly enhance gene silencing efficiency and inhibit tumor cells growth. In vivo experiments indicated that topical application of STAT3 siRNA PEI complex delivered by dissolving MNs into skin can effectively suppress the development of melanoma through silencing STAT3 gene, and the inhibition effect is dose-dependent. STAT3 siRNA delivery via dissolving MNs is a promising approach for skin melanoma treatment with targeting inhibition efficacy and minimal adverse effects.Hyperactivity of signal transducer and activity of transcription 3 (STAT3) plays a crucial role in melanoma invasion and metastasis. Gene therapy applying siRNA targeting STAT3 is a potential therapeutic strategy for melanoma. In this article, we first fabricated safe and novel dissolving microneedles (MNs) for topical application of STAT3 siRNA to enhance the skin penetration of siRNA and used polyethylenimine  as carrier to improve cellular uptake of siRNA. The results showed that MNs can effectively penetrate skin and rapidly dissolve in the skin.  It leads to ~75% deaths associated with skin cancer2. In recent years, the incidence of melanoma is dramatically increasing3. Melanoma is hard to cure and causes high mortality in the clinic due to poor prognosis, aggressive local growth, high rate of metastasis, and resistance to traditional radiotherapy4. The typical therapeutic options for melanoma include surgical resection, chemotherapy, immunotherapy, radiotherapy, and biotherapy5. However, the therapeutic efficacy of these approaches is restricted due to drug resistance, high toxicity, and poor selectivity8. Small interfering RNA (siRNA) therapy is a promising gene therapy for tumor with relative low toxicity and high specificity9. In most malignant tumors, signal transducer and activator of transcription 3 (STAT3) is related to malignant behaviors including tumor proliferation, metastasis, angiogenesis, survival, and immune evasion10, and it is hyperactive in many malignant cancers such as breast cancer, prostate cancer, and melanoma13. The activation of STAT3 can increase the code of apoptosis inhibition genes Bcl-xl, Bcl-2, Mcl-1, and survivin14, and improve the expression of cell cycle regulatory cyclin D1 and myc16. The tumor cell apoptosis is consequently inhibited. Moreover, activated STAT3 in melanoma can directly combine to matrix metalloproteinase-2 (MMP-2) gene promoter to increase the expression of MMP-2. MMP-2 degrades extracellular matrix, thus promoting the invasion and metastasis of tumor cells17. Finally, the activated STAT3 improves the expression of VEGF in melanoma to stimulate angiogenesis of tumor and enhance the tumor vascular permeability20. Overall, sustained abnormal activation of STAT3 can inhibit tumor cell apoptosis, promote tumor cell proliferation, and enhance angiogenesis22. Some studies on melanoma have shown that STAT3 increases the expression of antiapoptotic genes Bcl-xl, Mcl-1 and further promote melanoma proliferation and malignant transformation17. Moreover, hyperactivity of STAT3 in melanoma increases the expression of VEGF to induce angiogenesis24. Down-regulation of STAT3 can inhibit the melanoma development and metastasis24. Hence, STAT3 is a potential target for melanoma therapy25.Melanoma, a severe malignant tumor, initiates in melanocytes26. However, delivery of macromolecular siRNA into skin remains a challenge. Stratum corneum (SC), the outermost layer of skin, consists of dead cells (corneocytes) imbedded in lamellar extracellular lipid matrix, which presents a significant barrier to the delivery of exogenous agents27. siRNAs are hydrophilic with high molecular weight (~13 kDa) and negative charge. These properties hinder siRNAs from penetrating SC. Melanocytes exist in the basal epidermis and upper dermis of skin28. Hence, siRNAs require to be delivered not only across SC but also through viable epidermis with the aim to reach the target of melanocytes. Microneedles (MNs) have been shown to overcome SC barrier, penetrate the skin and into the viable epidermis or dermis in a minimally invasive manner29, and they have been used for the delivery of macromolecules including human growth hormone30, interferon-α-2b31, hepatitis B surface antigen32, insulin35, anti-PD1 antibody36, and siRNA38. Therefore, we hypothesized that STAT3 siRNA could effectively be delivered via MNs to the deep layer of skin, readily accumulate in the local tumor, targetedly silence STAT3 gene, and directly inhibit the melanoma development without leakage into the systemic circulation for reducing the toxicity.Currently, the application of siRNA to treat skin diseases such as psoriasis, melanoma tumors, and atopic dermatitis attracts serious attentions from scientistsin vitro, and the anti-melanoma activity of PEI/siRNA delivered by dissolving MNs was evaluated with mouse melanoma in vivo.With the aim to test the hypothesis of topical siRNA targeting STAT3 as a gene therapy strategy for melanoma, we developed for the first time a novel intradermal delivery system for STAT3 siRNA based on dissolving MNs. The dissolving MNs were fabricated with biocompatible vehicles of hyaluronic acid (HA), dextran, and polyvinylpyrrolidone (PVP) 17. STAT3 siRNA was encapsulated with polymeric carrier polyethylenimine , and then loaded within dissolving MNs. The cellular uptake, anti-proliferation, and gene silencing efficiency of STAT3 siRNA PEI complex (PEI/siRNA) were observed with B16F10 melanoma cells 2. When the MNs patch was observed under scanning electron microscope, the MNs presented the arrays comprised of 144 . As shown in Fig. To further assess the inhibition effect of STAT3 siRNA on B16F10 cells, 3- -2,5-diphenyl-2-H-tetrazolium bromide (MTT) was used to evaluate the cell viability. B16F10 cells proliferation was significantly inhibited when they were incubated with PEI/siRNA in comparison with the untreated group for 48 h , and administration protocol was assigned with Fig. P < 0.05 at time point of day 9, and 10, and P < 0.01 at time point of day 11 and 13), and at time of day 8, the tumor volume of G3 or G5 significant smaller than that of G1 (P < 0.05). However, there was no significant difference between G2, G3, and G4 from the time point of day 9 to the termination of day 13. Interestingly, the tumor volume of G5 was further significantly smaller than that of G2, G3, or G4 (P < 0.01). The results reveal that different administration protocols with the same total dose exert the similar inhibition effects on tumor development, and the anti-tumor effect is dose-dependent.To evaluate the antitumor effect of STAT3 siRNA on melanoma P < 0.01, Fig. P < 0.01), and about one fifth of that of G1. Based on the results from tumor volume and weight, it can be concluded that the topical delivery of PEI/siRNA via dissolving MNs can inhibit the tumor growth of melanoma, and the inhibition effect is dose-dependent.At the time point of termination (day 13), the tumor was isolated and weighted. The tumor weight of G2, G3, or G4 was significantly lower than that of G1, and there was no significant difference between G2, G3, and G4. However, the relative mRNA expression level of G5 was significantly lower than that of G2, G3, or G4 (P < 0.01), and it was reduced to 70%. Consistent with the inhibition effects of PEI/siRNA on tumor volume and weight, the gene silencing effects of PEI/siRNA on tumor tissue was confirmed in vivo, and the silencing effect was also dose-dependent. The gene silencing effects of PEI/siRNA on STAT3 mRNA expression in vivo was the antitumor molecular mechanism of STAT3 siRNA against melanoma.After the termination of the treatment experiment, tumor tissues from different groups were isolated and total RNA was extracted. RT-PCR was used to measure STAT3 mRNA expression. As shown in Fig. in vivo, and the inhibition effect is dose-dependent.Based on the histological analysis Fig. , tumor o42. Currently, new siRNA-based medicines and siRNA therapy have been developed in clinical trials43. Malignant tumors are genetic diseases involved a line of cumulative epigenetic and mutational changes which cause uncontrollable cell metastasis, differentiation and proliferation45. The major advantage of siRNA technology is specificity by the principle of Watson-Crick base pairing to discriminate target gene versus non-target gene46. A series of preclinical researches have indicated that gene silencing could suppress tumor development involving tumor cell apoptosis, proliferation, angiogenesis, metastasis, immune escape, and chemo-resistance49. STAT3 is hyperactive in melanoma, its abnormal activation promotes tumor cell proliferation, suppresses apoptosis, and induces invasion and metastasis; therefore, silencing STAT3 gene by siRNA is a promising strategy for treating melanoma51. However, applying siRNA gene therapy for skin melanoma, which initiates in the basal layer of epidermis and upper dermis in vivo, faces two obstacles: delivery and transfection. While intradermal delivery technology breaches the barrier function of SC, further carrier is required to facilitate cellular uptake of siRNA. The goal of our study was to design a dissolving microneedle system to deliver STAT3 siRNA into the skin in a targeted manner to breach the stratum corneum and directly reach the site of basal layer of epidermis and upper dermis, and PEI was used as the carrier to increase the transfection efficiency. It is evident that siRNA can form tight complex with the cationic PEI via attractive electrostatic interactions and be internalized by cells with the help of cationic complex52. PEI /siRNA of our current study can effectively facilitate siRNA uptake by B16F10 cells, the percentage of FAM positive B16F10 cells approached ~100% after incubation with PEI/FAM-siRNA for 4 h, which was significantly higher than that of incubation with naked siRNA (~1%), and the complex down-regulated STAT3 mRNA to 60.4%, B16F10 viability of PEI/STAT3 siRNA treated decreased by 33.6%, while the gene expression and cell viability of B16F10 cells treated with naked STAT3 siRNA almost remained the similar to those of non-treated B16F10 cells. These results were consistent with previous findings that naked STAT3 siRNA presented no significant inhibition on B16F10 cells growth51. STAT3 siRNA can silence STAT3 gene of B16F10 cells; however, it must be first transferred into B16F10 cells via carrier due to its own poor cellular permeability, and PEI is an effective vehicle for transferring STAT3 siRNA into B16F10 cells. Development of efficient and safe carrier to transfer STAT3 siRNA into cells will be critical for its clinical application, which will be the subject of our further research.In recent years, gene silencing technology has developed rapidly and become a potential therapy for human illnesses. Among which, siRNA therapy is the most hopeful and has received widespread attention29. MNs presented a promise for intradermal delivery of macromolecules, and coated steel MNs53, hollow MNs54, and solid MNs38 have been reported to deliver siRNA into the skin for treating skin disorder caused by aberrant gene expression. Compared to the other MNs , dissolving MNs can dissolve in the skin interstitial fluid of viable epidermis/dermis within minutes and release the drug payload, avoiding biohazardous sharp waste in the skin and reducing any risk of infection transmission56.MNs, a minimally-invasive, patient-friendly, and self-administered approach, can overcome SC barrier and deliver the payload to viable epidermis and dermis, and modulation of MNs geometry and formulations can result in controlled drug deposition within targeted skin layersin vivo and subsequent anti-melanoma and gene silencing efficacy. The designed dissolving MNs based on dextran, HA, and PVP with a ratio of 4:1:1 (w/w/w) possessed mechanical strength of ~86 N, and almost all the microneedle arrays in a given external force of 20 N punctured the skin and remained in shape in the skin. The depth of insertion was ~330 μm, which ensured delivering the siRNA loaded into the epidermal basal layer and upper dermis, where melanoma cells were located. However, MNs cannot penetrate the skin to their full length (~650μm), which is consistent with previous reports57. Therefore, we optimized the drug loading method to make siRNA preferentially located toward the upper tip of MNs, thereby enabling complete siRNA delivery even with incomplete microneedle penetration. The CLSM images before and after administration of MNs showed that MNs fully dissolved within 5 min after puncturing the skin. We therefore expect that the MNs can dissolve in the skin upon contacting the interstitial fluid of viable epidermis/dermis within minutes and release and deposit the siRNA loaded into the skin.The study is the first to explore the potential of dissolving MNs for delivering STAT3 siRNA into skin 3vs 704.77 ± 49.36 mm3, 0.16 ± 0.02 g vs 0.80 ± 0.05 g), while being treated with total dose of 132 µg of STAT3 siRNA, the tumor volume and weight decreased by ~50% . However, different administration protocols with the same total dose of STAT3 siRNA exert the similar inhibition effects on tumor development. The tumor necrosis observed from histological analysis further confirmed the anti-tumor results. Moreover, there was no significant difference of the body weight of tumor-bearing mice with different treatment protocols and doses, which might be attributed to the targeting delivery of STAT3 siRNA to melanoma via dissolving MNs and the selective effect of STAT3 siRNA on STAT3 gene of melanoma cells. Consistent with in vitro, PEI/siRNA showed an effective down regulation of STAT3 mRNA expression in vivo, which was the antitumor molecular mechanism of STAT3 siRNA against melanoma. We demonstrated for the first time that dissolving microneedle is an effective and safe strategy for targeting delivery of STAT3 siRNA via skin to melanoma cells and exerts specific anti-melanoma effect.As expected, the dissolving microneedle effectively delivered PEI/siRNA topically to B16F10 melanoma tumor-bearing mice and exerted the tumor inhibition efficacy of siRNA, and the anti-tumor effect was dose-dependent on siRNA. After being treated with total dose of 264 µg of STAT3 siRNA, the tumor volume and weight decreased by ~80% compared with the untreated animal  and Sprague Dawley (SD) rats (200–220 g) were obtained from Experimental Animal Center of Guangzhou University of Chinese traditional medicine (License number: SCXK (YUE) 2013–0034). All protocols for animals were in accordance with the Institutional Animal Care and Use of Sun Yat-sen University.All experimental protocols were approved by the Animal Experimentation Ethics Committee of Sun Yat-sen University.2.Mouse melanoma cell line B16F10 was purchased from American Type Culture Collection  and incubated in RPMI-1640 medium supplemented with 10% FBS at 37 °C with 5% COThe siRNA targeting murine STAT3 mRNA, negative control siRNA, FAM-labeled siRNA, and STAT3 primers were synthesized by Gene Pharm . β-actin primers were bought from Sangon Biotech . The sequence is: STAT3 siRNA: sense: 5′-GGACGACUUUGAUUUCAACtt-3′; antisense: 5′-GUUGAAAUCAAAGUCGUCCtg-3′. Negative control siRNA: sense:5′-UUCUCCGAACGUGUCACGUTT-3′; antisense: 5′-ACGUGACACGUUCGGAGAATT-3′. β-actin primer forward: 5′-CCAACCGCGAGAAGATGA-3′; reverse: 5′-CCAGAGGCGTACAGGGATAG-3′.siRNA was added to the same volume of PEI solution at the N/P ratio of 10:1, and the mixture was incubated at room temperature for 30 min to obtain PEI/siRNA complex. The diameter and potential of the complex determined with Dynamic Light Scattering  were 135.1 ± 5.2 nm and 34.6 ± 2.2 mV, respectively.A stainless pyramidal mould of 144 (12 × 12) MNs with height of 650 μm and base of 300 μm was used as the master mould. The inverse mould was prepared by casting polydimethylsiloxane (PDMS) over the master mould and degassed in a vacuum desiccator for 1 h followed by curing in the 95 °C oven for 30 min to solidify the polymer. Then the PDMS mould was obtained after peeling off the master mould and repeatedly used to fabricate dissolving MNs.A two-step moulding process was applied to prepare drug-loaded dissolving MNs. Firstly, a mixture of dextran 40, PVP 17, and HA at a weight ratio of 4:1:1 was dissolved in ultrapure water, and then Rh-B, PEI/STAT3 siRNA, or PEI/FAM-siRNA was added and mixed uniformly to obtain drug-loaded needle solution. Fifty milligrams drug-loaded needle solution was dipped into PDMS mould, and the mould was degassed under vacuum for 10 min and then centrifuged at 4000 rpm for 20 min so that the needle solution filled the PDMS mould microcavities completely, obtaining the needle tip layer. Secondly, a mixture of dextran 40, PVP 17, and HA at a weight ratio of 3:3:1 was dissolved in ultrapure water to obtain the base solution. One hundred fifty milligrams of base solution without drug was casted onto the tip layer surface, then centrifuged at 4000 rpm for 10 min and dried for 24 h at 4 °C. Eventually, the drug-loaded dissolving MNs was obtained by peeling off from PDMS mould. The dissolving MNs loaded with Rh-B, PEI/STAT3 siRNA, or PEI/FAM-siRNA were respectively characterized with digital camera , scanning electron microscope , and confocal laser scanning microscope . Rh-B, PEI/STAT3 siRNA, or PEI/FAM-siRNA MNs were stored in 4 °C and away from light.A TA.XT plus texture analyzer  was used to determine the mechanical properties of PEI/siRNA loaded MNs under an axiload. Stress versus strain curves were generated with force and displacement. The test station pressed MNs against a rigid metal surface at a rate of 1.1 mm/s. The texture analyzer recorded the force until a preset displacement of 600 μm was reached.To measure the insertion efficiency of dissolving MNs, excised SD rat skin was used as skin model. The back skin was shaved, excised, subcutaneous fat removed, and cleaned with PBS and ethanol; then the skin was stretched on Styrofoam and fixed with pins. The microneedle patch was attached to the removable cylindrical probe of texture analyzer with double sides adhesive tape. The microneedle patch following the probe inserted the skin at a rate of 1.1 mm/s with different forces of 10 N, 20 N, and 30 N, respectively, and retained in the skin for 60 s. After the MNs were removed from the skin, trypan blue solution  was added onto the punctured skin and stayed for 10 min, trypan blue would penetrate through the pinholes. After washing off the trypan blue solution, the stained pinholes in the skin were calculated.The depth of MNs insertion into the skin was measured by optical coherence tomography  . The rat skin was treated as above described and placed on a PDMS substrate for support. MNs inserted the skin with a force of 20 N by thumb pressing. Then the depth of insertion was observed with OCT in real time. The OCT parameters were set as follows: low-coherence light with power of 5 mW, full width at half maximum (FWHM) of 45 nm, laser center wavelength at 830 nm, and scanning rate at 18 kHz B-scans. Matlab Version 7.14  was used to image the skin layers and MNs.PEI/FAM-siRNA loaded MNs inserted the rat skin for 0, 1, 3 or 5 min respectively and then were removed. CLSM was used to observe the residual length of MNs.CLSM. CLSM was used to visualize the uptake of FAM-labeled siRNA by B16F10 cells. B16F10 cells of 1 × 105 were cultured in dishes. PEI/FAM-siRNA or naked FAM-siRNA were added at a final siRNA concentration of 100 nM and incubated with cells for 4 h. Afterwards, the cells were washed with PBS for four times and fixed with 4% paraformaldehyde for 30 min. DAPI was used for dyeing cell nucleus. Finally, the samples were visualized under CLSM. He Ne laser was used to excite FAM at 488 nm.For quantitative evaluation of transfection efficiency, the cellular uptake of FAM-siRNA was measured by flow cytometry. After B16F10 cells was transfected with PEI/FAM-siRNA or naked FAM-siRNA at a final siRNA concentration of 100 nM as above described, the percentage of FAM positive B16F10 cells and average intensity of fluorescence were measured with flow cytometry.TM RT reagent kit with gDNA eraser . Reverse Transcription System  was used to synthesize cDNA, and SYBR Green was employed to real-time PCR. LightCycler480 II  was used to measure the threshold cycle number (Ct) of STAT3 and β-actin. STAT3 and β-actin mRNA was calculated by 2−ΔΔ Ct relative-quantitative method.RT-PCR was performed to confirm the silencing efficiency of STAT3 siRNA on STAT3 gene of B16F10 cells. After B16F10 cells were transfected for 4 h with naked STAT3 siRNA and PEI/STAT3 siRNA respectively at a final siRNA concentration of 100 nM. The residual uninternalized complex was removed with RPMI 1640 and the cells were continued to incubate for 24 h. Afterwards, the cells were washed with PBS for three times, and trizol was added to extract total RNA following the instruction protocol. Total RNA of 500 ng as loading control was treated with PrimeScript3 B16F10 cells were cultured in 96-well plates overnight, then the cells were transfected with PEI, PEI/siRNA or naked STAT3 siRNA (100 nM siRNA per well), respectively. After 4 h transfection, the uninternalized complexes was removed with RPMI 1640 and the cells were continued to incubate for 48 h. Cell proliferation activity was measured by adding 20 μL of MTT per well. After cultivation for 4 h, 150 μL of DMSO was added to each well to dissolve formazan crystals. The optical density (OD) value was detected at 490 nm using FlexStation III Multi-Mode Microplate Reader .Cell growth was measured with MTT cell proliferation assay. Briefly, 5 × 106 B16F10 cells in the left flank, and melanoma reached 50–60 mm3 on day 8 after inoculation based on our preliminary experiment. After inoculation, the mice were treated as follows: 1) Group 1 (G1) was the tumor model control group without administration; 2) Group 2 (G2), each mouse was administered with PEI/siRNA of 33 μg per day via MNs on day 8 after inoculation and for 4 consecutive days . Each mouse was subcutaneously injected with 1 × 10ays Fig. ; 3) Grouays Fig. ; 4) Grouays Fig. ; 5) Grouays Fig. . TherefoEight days after inoculation tumor cells, tumor size of each mouse was detected with digital caliper over time. The tumor volume was calculated by the following formula:Moreover, mouse body weight was monitored daily to evaluate the potential toxicities. At the time point of day 13, the mice were euthanized. Tumors were immediately isolated, washed with PBS to remove blood, dried with tissue paper, and weighted. The tumor tissue was sectioned, fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin. Slice of 3 μm tumor tissue was stained with standard HE procedure. Slices were observed with optical microscope  for histological analysis.in vivo, the clean tumor tissue was treated with trizol solution and homogenized to form uniform cell suspension by ultrasonic cell disruptor . Subsequently, total RNA was extracted and analyzed with RT-PCR as mentioned in vitro.To assess gene silencing effects All measurements were performed at least in triplicate. The data presented as mean ± standard deviation (SD). The statistical difference was compared with one-way analysis of variance (ANOVA) followed by Least Significant Difference (LSD) as post hoc analysis . Statistical significance was set at P < 0.05.The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary information


This study tested whether including objects perceived as highly interesting by children with autism during a gaze following task would result in increased first fixation durations on the target objects. It has previously been found that autistic children differentiate less between an object another person attends to and unattended objects in terms of this measure. Less differentiation between attended and unattended objects in ASD as compared to control children was found in a baseline condition, but not in the high interest condition. However, typically developing children differentiated less between attended and unattended objects in the high interest condition than in the baseline condition, possibly reflecting reduced influence of gaze cues on object processing when objects themselves are highly interesting. We are constantly surrounded by multiple stimuli competing for our attention. When interacting socially, other people’s gaze can constitute important cues as to what stimuli are currently important to attend to. Acknowledging what our interaction partner is looking at often provides information as to what is going on in that person’s head, and thus facilitates interaction as well as social learning. The ability to follow other people’s gaze therefore serves an important function. Gaze following is a crucial aspect of joint attention—the sharing of attention between two individuals and an object  linked to an event of interest. In reading research, first fixation duration is believed to reflect the initial lexical activation process Rayner , and it Preliminary evidence suggests that the first fixation duration measure can reveal the extent to which an individual takes contextual cues such as another’s gaze into account during object processing, and that the measure can discriminate between children with ASD and controls , where the stimuli were presented. A 5-point calibration was conducted before the start of the stimuli presentation, and repeated if needed. The stimulus videos were intermixed with other stimuli and attention grabbers and presented in three blocks, with short breaks in between. The order of the videos was pseudo-randomized so that no gaze following clips occurred directly after each other, and no more than two clips from the same condition occurred in a row. The WPPSI-III/WISC-IV and the ADOS-2 (ASD group only) were administered at the same visit as the eye tracking. The SRS and RBS-R were completed by the parents before the visit.The stimuli were 16 videos . In accordance with our previous study . did follow gaze, only data from trials with congruent gaze shifts was included in this analysis. We chose not to use a difference score as in our previous paper . Three dependent measures were defined as follows: (1) Accuracy was calculated as a proportion where the number of congruent gaze shifts was divided by the total number of (congruent and incongruent) gaze shifts. Because trials where the child looked at the object next to the attended object first were removed from the analyses see “” sectiont(15) = 8.24, p < 0.001; TD: t(16) = 6.05, p < 0.001) and the baseline condition (ASD: t(15) = 6.08, p < 0.001; TD: t(16) = 5.11, p < 0.001; one sample t-tests comparing the means to 0.33). A repeated measures ANOVA on the accuracy scores revealed no main effect of condition, F = 0.10, p = 0.756, partial η2 = 0.003, or group, F = 2.49, p = 0.125, partial η2 = 0.07, and no interaction effect between group and condition, F = 0.02, p = 0.878, partial η2 = 0.001. For descriptive statistics, see Table The gaze following accuracy of both groups was, as expected, greater than chance in both the high interest condition  = 11.31, p = 0.002, partial η2 = 0.27, but no main effect of condition, F = 0.13, p = 0.721, partial η2 = 0.004, or group, F = 0.86, p = 0.362, partial η2 = 0.03  = 2.95, p = 0.024, d = 1.06. Moreover, as expected, no difference between groups was found in terms of the AUF-index when the objects belonged to such categories (high interest condition), t(31) = 1.47, p = 0.608, d = 0.53 (independent samples t-tests). Contrary to our expectations however, no difference between conditions was found in the ASD group, t(15) = 1.65, p = 0.476, d = 0.85, but in the TD group a significantly higher AUF-index was found in the baseline as compared to high interest condition, t(16) = 4.06, p = 0.004, d = 2.03 (paired samples t-tests). For descriptive statistics, see Table To test our main hypothesis, namely whether the two groups were affected differently by the object manipulation in terms of the AUF-index, a repeated measures ANOVA with condition as within subjects variable and group as between subjects variable was performed on this measure. The analysis revealed a group by condition interaction effect, see Fig. , a higheF1, 1 = 11.31F = 11.77 p = 0.002, partial η2 = 0.28, with more looking time at high interest compared to baseline objects  = 0.64, p = 0.429, partial η2 = 0.02, and no interaction effect between group and condition, F = 0.38, p = 0.540, partial η2 = 0.01, was found. The analysis thus showed that the children, regardless of group status, looked more at the objects in the high interest condition than in the baseline condition.A repeated measures ANOVA with total looking time as the dependent measure, condition as within subjects factor and group as between subjects factor revealed a main effect of condition, F1, 1 = 11.77t(31) = 1.74, p = 0.092, d = 0.63 or the baseline condition, t(31) = 1.30, p = 0.204, d = 0.47 (independent samples t-tests). The number of valid trials in the first fixation duration analysis also did not differ between groups in either the high interest condition, t(31) = 0.03, p = 0.973, d = 0.01 or the baseline condition, t(31) = 0.02, p = 0.988, d < 0.01. For descriptive statistics, see Tables The groups did not differ in terms of the number of valid trials in the accuracy analysis in either the high interest condition, t1 = 1.74,F = 0.57, p = 0.455, partial η2 = 0.02, or group, F = 0.08, p = 0.775, partial η2 = 0.003, and no interaction between group and condition, F = 0.22, p = 0.643, partial η2 = 0.007.To evaluate whether there were general differences in first fixation durations between groups or objects types, a separate analysis was conducted on the mean first fixation duration on all objects and trials, irrespective of gaze following performance. A repeated measures ANOVA revealed no main effects of condition, r = 0.25, p = 0.408; TD: r = 0.20, p = 0.442) or the baseline condition . Neither were any significant correlations between AUF-index and any ADOS-2, SRS or RBS-R scores found.No correlations between AUF-index and NVIQ were found in either the high interest condition  = 7.00, p = 0.014, partial η2 = 0.22, and no interaction effects between condition and age, F = 0.01, p = 0.942, partial η2 < 0.001, or NVIQ, F = 0.07, p = 0.798, partial η2 < 0.01 were found. Note that the N in the ASD-group for this analysis is 13, since it was not possible to establish NVIQ in three of the children.Finally, the main AUF-index analysis was rerun with age and non-verbal IQ entered as covariates. The group by condition interaction effect remained significant, higher gaze following accuracy in the ASD group than TD group. The present results thus add to the converging evidence showing that in a laboratory based task with a model using both head and eyes as cues, children with or at risk for ASD are as likely to follow gaze spontaneously as typically developing children  humans could also be included. Greater care should also be taken to control for low level features that may differ between the objects making up the different conditions.In conclusion, the current results validate the AUF-index as a measure for detecting subtle differences in gaze following behavior (see Falck-Ytter et al.


Results showed early-passage DP cells strongly expressed miRNAs related to inhibition of noncanonical Wnt pathways. In late-passage DP cells, miRNAs capable of inhibiting the canonical Wnt/β-catenin pathway were upregulated, in addition to the miRNAs targeting the noncanonical Wnt pathway. Moreover, we verified that β-catenin expression was downregulated by miR-195-5p overexpression in dose manner. Meanwhile LRP6 expression was downregulated in both protein and mRNA as well as the genes involved in the hair inductivity of DP cells. These results suggest that the appearance of miRNAs that suppress the Wnt/β-catenin pathway may be responsible for the loss of ability of DP cells in culture and miR-195-5p is the potential key factor involved in regulating HF inductivity of DP cells.Dermal papilla (DP) cells play a vital role in hair follicle (HF) development and postnatal hair cycling. However, the abilities are lost on further culture. Recent studies have demonstrated significant influences of posttranscriptional regulation by microRNA (miRNA) on HF development. The current study aims to investigate how miRNAs regulate Wnt/ Cultured DP cells can induce hair follicle differentiation in epithelial cells and are required for hair reconstitution to alleviate baldness –3. DP ceβ-catenin pathway activates genes, including CCN1, CCN2 [ OVOL1 [ Versican [β-catenin signaling activation such as a GSK-3β inhibitor, 6-bromoindirubin-3′-oxime (BIO), and Wnt3a [β-catenin gene within the DP cells of fully developed hair follicles results in premature induction of the destructive phase of the hair cycle (catagen) and also prevents stem cells resident in the permanent portion of the follicle from regeneration of the cycling follicle [β-catenin activity in the dermal papilla regulates at least two (FGF and IGF) signaling pathways that mediate the inductive effects of the DP on keratinocytes [The Wnt/N1, CCN2 , OVOL1 [ [ OVOL1 , and VerVersican , which aVersican –18: (1) nd Wnt3a , (2) actfollicle , and (3)MicroRNAs (miRNAs), the small noncoding RNAs of approximately 22 nucleotides, have been implicated in regulating a number of signaling pathways, including the Wnt pathway . FurtherHowever, the expression profile of miRNAs and potential role in regulating Wnt or related signaling pathway in different passage DP cells are unknown. Consequently, in this study we aim to investigate the expression pattern of miRNAs in early-passage and late-passage DP cell samples and their putative role with mechanism in HF inductivity.2 at 37°C. The fourth and tenth DP cells were chosen to represent early-passage and late-passage DP cells, and functional study of their different hair follicle inductivity abilities has been implemented in our previous work [Three human scalp specimens were obtained from one female and two male patients  undergoing selected face-lift surgeries. Primary culture and subculture of DP cells were performed as previously described . DP cell2 plastic flask after reaching confluence, at a density of 6 × 107 and 6 × 106 per flask, respectively. The samples were labeled as follows: DP4-a, DP4-b, DP4-c, DP10-a, DP10-b, and DP10-c. Total RNA was extracted from human DP cell samples using Trizol reagent (Invitrogen) and an miRNeasy mini kit (QIAGEN) according to manufacturer's instructions. RNA quality and quantity were measured by using a Nanodrop spectrophotometer  and RNA integrity was determined by gel electrophoresis.Each passage-4 and passage-10 DP cell sample derived from the above three specimens was collected from a 75 cmP value ≤ 0.05.DP cell miRNA profiles were obtained by microarray analysis performed by KangChen Bio-tech and using Exiqon Array platforms. The miRCURY LNA™ microRNA Array contains 3100 capture probes, covering all human, mouse, and rat miRNAs annotated in miRBase 18.0. Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Expressed data were normalized using the median. After normalization, differentially expressed miRNAs were identified through volcano plot filtering. Hierarchical clustering was performed using MEV software . The threshold value we used to screen up- and downregulated miRNAs was a fold change ≥ 1.50 and a −∆∆CT) method. Primer sequences used for amplification are listed in miRNA Q-PCR was performed using a PCR master mix (Superarray) according to the manufacturer's instructions. U6 was used as the internal control. Thermal cycling conditions were as follows: 10 min at 95°C and 40 cycles of 10 s at 95°C and 60 s at 60°C. Data were analyzed by the relative quantification (2http://www.geneontology.org). The pathway is based on the latest Kyoto Encyclopedia of Genes and Genomes  database.TargetScan, miRBase, and miRanda miRNA target databases were used to identify putative miRNA targets. Gene ontology (GO) analysis is a functional analysis associating target genes of each microRNA. The ontology covers three domains: biological processes, cellular components, and molecular function. The GO categories are derived from Gene Ontology , respectively, using Lipofectamine 2000  following the manufacturer's instructions. Then 30 nM specific miRNA mimic and control oligos were transfected into the cells. All of the above-mentioned miRNA mimics/inhibitors were commercially synthesized by Ambion (USA).β-catenin (1 : 1000), anti-Wnt3A (1 : 1000), anti-Wnt7A (1 : 1000), anti-Noggin (1 : 1000), or anti-Wnt5A (1 : 1000) antibodies  overnight at 4°C and then washed and incubated for 1 h with secondary antibodies .Cultured cells were lysed in RIPA buffer, and lysates were analyzed using a standard western blot procedure. GAPDH was used as the loading control. PVDF membranes were incubated with anti-GAPDH (1 : 5000), anti-LRP6 (1 : 1000), anti- t-test. Data are expressed as the mean ± SD. A P value of <0.05 was considered statistically significant. All experiments were repeated at least three times, and for each experiment samples were analyzed in triplicate.The statistical significance of the differences was determined using Student'shttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE77825). Of the total 1924 hsa-miRNAs (miRNAs) screened  (Our previous study demonstrated that the follicle-inducing ability of cultured DP cells declined with passage . To delie > 1.5) . Based oe > 1.5) . We perfe > 1.5) .P < 0.05)  .miRNAs regulate a large number of target genes, and several databases based on various algorithms are available for predicting miRNA gene targets. We chose TargetScan, miRanda, and miRBase to predict gene targets of the 133 differentially expressed miRNAs. This resulted in the identification of 550 gene targets for upregulated miRNAs , intracellular components (ontology: cellular component), and protein binding (ontology: molecular function) and that the most highly enriched GOs targeted by the downregulated transcripts involved biological regulation , intracellular components (ontology: cellular component), and protein binding (ontology: molecular function) (Supplementary Homo sapiens (human),” which corresponded to downregulated miRNAs targeting 259 genes. Notably, Wnt signaling necessary for the follicle development was included in pathways targeted by both up- and downregulated miRNA  cells to regulate Wnt signaling and β-catenin transcriptional activity. Because of the Wnt signaling which was one of the key regulatory pathways for the follicle inductivity of dermal papilla, we hypothesized that the changes in miR-335-5p and miR-195-5p expression interfere with Wnt signaling. To test this hypothesis, we characterized the expression of β-catenin, which is upregulated in active Wnt signaling in early-passage DP cells. Due to the weak state of passage-10 DP cells, we altered the levels of miRNA only in passage-4 DP cells. Passage-4 DP cells were transfected with various concentrations of either an miR-335-5p inhibitor or an miR-195-5p mimic for 72 and 96 hours  at 72 and 96 hours. However, transfection of DP4 cells with the miR-335-5p inhibitor did not significantly downregulate β-catenin expression. Therefore, the decline in ability of DP cells to induce hair follicle development could be due to the overexpression of miR-195-5p, observed in DP10 cells, resulting in the reduction of β-catenin in the cytoplasm and a decline in Wnt signaling.In the differentially expressed miRNA profile, miR-335-5p and miR-195-5p, previously reported to regulate Wnt/ Figures . Resultsβ-catenin levels  and DP cells incapable of (passage 10) hair follicle induction. We identified 28 genes involved in Wnt signaling and 3 genes involved in FGF and BMP signaling, suggesting a large effect of miRNA on Wnt signaling. In late-passage DP cells, miRNAs that were overexpressed were predicted to inhibit canonical Wnt signaling, whereas in early-passage DP cells the miRNAs that could activate Wnt/egulated . This inβ-catenin canonical signaling. A recent report suggests that under normal circumstances the bone marrow microenvironment is more conducive to noncanonical Wnt signaling, compared to Wnt/β-catenin canonical signaling, in order to maintain the resting state of hematopoietic stem cells [The role of noncanonical Wnt signaling function in DP cells is not yet clear. Because of Wnt competition for its receptors, noncanonical Wnt signaling would inhibit Wnt/em cells . As the em cells . This stβ-catenin signaling plays a vital role in hair follicle regeneration in DP cell transplantation [β-catenin in DP cells. Thus, miR-195-5p downregulates Wnt/β-catenin signaling and may be one of the reasons of late-passage DP cells losing the ability to induce hair follicles.Many studies have shown that Wnt/antation . It has In this study, we predicted that Wnt3a, Wnt7a, and LRP6 were miR-195-5p target genes according to the three bioinformatic algorithms above. Of the three, only LRP6 expression is affected by miR-195-5p. This contrasts a previous study that Wnt3a is directly targeted by miR-195-5p . The difβ-Catenin interacts with coregulators of transcription factors, including LEF-1, to activate Wnt/β-catenin-dependent gene transcription [cription . LEF-1 mcription . In DP lcription . The resβ-catenin, LEF-1, Wnt5a, and Noggin, which are involved in Wnt, SHH, Notch, and BMP signaling (Wnt5a and Noggin are DP cell markers for hair follicle-inductive properties , 41. Wntignaling . Our resβ-catenin activation by targeting LRP6 and signature genes of Wnt signaling, indicating its potential involvement in HF inductivity regulation. Further functional experiments are required to understand the related mechanism.The present study found expression profile of miRNAs in early- and late-passage DP cells and elucidated that upregulation of miR-195-5p could inhibit Wnt/