Aging is a major risk factor for morbidity and mortality from cardiovascular causes in men. To better understand the cellular processes related to age-related cardiac complications, we undertook research aimed at comparative evaluation of genes expression and distribution of β-catenin, CacyBP/SIP, galectin-3 and LMP7 in the heart of healthy men in different age groups. The study was conducted on the hearts of 12 men (organ donors) without a history of cardiovascular disease, who were divided into two age groups: men under and men over 45 years of age. On paraffin sections, immunohistochemical reactions were performed to detect β-catenin, CacyBP/SIP, galectin-3 and immunoproteasome subunit LMP7. The expression of genes coding β-catenin, CacyBP/SIP, galectin-3 and LMP7 was also evaluated by real-time PCR method. In the heart of men over 45 years old, both gene expression and immunoreactivity of β-catenin, CacyBP/SIP, galectin-3 and LMP7 were stronger compared to younger individuals. The results of the presented studies suggest that β-catenin, CacyBP/SIP, galectin-3 and immunoproteasomes might be involved in the internal regulation of heart homeostasis during ageing. One aspect of aging of the entire human body is the aging of the heart and circulatory system. As the aging process progresses, the structure and function of the heart changes .Despite continuous efforts by clinicians and scientists to increase the effectiveness of prophylaxis and treatment of cardiovascular complications in older people, cardiovascular diseases remain the main cause of mortality, accounting for over one third of all deaths . A more in vivo and in vitro studies have shown that β-catenin affects the differentiation and survival of myocardial cells [The β-catenin is a double-function protein in the cell. Cytoplasmic β-catenin determines cell function by modulating gene transcription. In contrast, membrane bound β-catenin coordinates cellular connections and is responsible for maintaining tissue architecture . This pral cells –5. β-catal cells –9. β-catal cells , 10–13.As a transcription factor, β-catenin is tightly regulated by proteasomal degradation. In the classical pathway, β-catenin is phosphorylated by a dual-kinase mechanism involving casein kinase I (CKI) and glycogen synthase kinase 3β (GSK-3β) in the so-called β-catenin destruction complex. Subsequently phosphorylated β-catenin is ubiquitinated and undergoes proteasomal degradation , 15. RecTo date, only few reports on the importance of CacyBP/SIP in the functioning of the cardiovascular system have been published and hence the current body of knowledge in this field is limited. Research on neonatal rats and cultured cardiac myoblasts (H9C2 cells) has demonstrated that CacyBP/SIP plays an important role in the differentiation of cardiomyocytes and heart development . The samRecent literature reports indicated that β-catenin signalling is also regulated by galectin-3. Galectin-3 is a member of the galectin family, which comprises the peptides having at least one carbohydrate recognition domain and specific affinity for β-galactosides . It has Galectin-3 is now considered one of the prognostic markers of heart failure and death from cardiovascular causes –25. IncrCellular senescence is accompanied by the impairment of numerous internal processes including disturbance in the balance between the synthesis and degradation of proteins in cells , 29. OneWith age, the activity of proteasomes decreased, which leads to the accumulation of damaged or non-functional proteins in cells , 29. TheTo date, only the effect of aging on the content of β-catenin and proteasomal activity in the heart of laboratory animals has been investigated , 33–35 aConsidering the involvement of β-catenin, CacyBP/SIP and galectin-3 in the regulation of cardiac function, as well as the strict functional dependence of these proteins on proteasomal activity, the current study was performed to evaluate immunohistochemically and to compare gene expression of β-catenin, CacyBP / SIP, galectin-3 and immunoproteasome subunit LMP7 in the heart of healthy men at different ages.2. The men were divided into two groups: subjects older than 45 years (six men) and subjects under 45 years old (six men).Twelve adult men (organ donors) without history of cardiovascular disease were used in this study. The men were in age range from 21–65 years, mean body weight 84.5 ± 1.26 kg and mean BMI (body mass index) 26.3 ± 0.28 kg/mEach man presented with clinical symptoms of brain death was considered to be an organ donor. Irreversible brain damage was confirmed by special clinical examination and angiography (no blood flow within the brain arteries).After diagnosis and confirmation of brain death and after collection of organs for transplantation , a heart sample was taken from each body and immediately fixed in 10% buffered formalin solution and routinely embedded in paraffin. Sections (4 μm) were stained with haematoxylin-eosin for general histological examination and processed by immunohistochemistry for detection of the β-catenin, CacyBP/SIP, galectin-3 and immunoproteasome subunit LMP7.The study protocol was approved by the Ethics Committee, Medical University of Białystok (R-I-002/345/2007), and a written informed consent had previously been obtained from each man or from his family member(s).™ EnVision™ Detection System, Peroxidase/DAB, Rabbit/Mouse detection kit  [Paraffin blocks were cut into 4-μm sections (3 sections from each subject for each antibody) and attached to positively charged glass slides. Immunohistochemistry was performed, using an REALomation) . Immunosomation) ), galect’-diaminobenzidine (DAB) substrate chromogen. The sections were finally counterstained in QS haematoxylin , mounted and evaluated under light microscope. Specificity test, performed for the β-catenin, CacyBP/SIP, galectin-3 and LMP7 antibody included a negative and positive control. In negative control antibodies were replaced by normal rabbit serum  at respective dilution. For negative control, no immunostaining was observed in heart tissues under the omission of the primary antibodies. Human tissues were a positive control: large intestine for β-catenin and galectin-3, prostate for CacyBP/SIP, and spleen for LMP7 , in proportion 1:4000 for β-catenin, 1:50 for CacyBP/SIP, 1:1000 for galectin-3 and 1:5000 for LMP7. The procedure was followed by incubation with secondary antibody (conjugated to horseradish peroxidase-labelled polymer). Appropriate washes with S 3006 Wash Buffer (DakoCytomation) were performed between each step. The bound antibodies were visualized by 1 minute incubation with liquid 3,3for LMP7 .Histological preparations were subjected to a visual analysis using an Olympus BX41 light microscope  with Olympus DP12 digital camera  and a PC computer and documented.2, 200x magnification (20x lens and 10x eyepiece)) from each segment of the heart. Each obtained digital image of the heart cross-section was subjected to morphometric evaluation using NIS Elements AR 3.10 Nikon software for microscopic image analysis. In each analysed image, all cardiomyocytes were counted (only those cardiomyocytes with visible nucleus were considered); then the number of cardiomyocyte cells was converted and presented as mean values per 1 mm2 section area.Fifteen (15) heart sections  were examined from each man. Five randomly selected microscopic fields , previously fixed in 10% buffered formalin solution and routinely embedded in paraffin, was taken from each patient. Total RNA was isolated using the High Pure FFPET RNA Isolation Kit (Roche) for formalin-fixed and paraffin-embedded tissues. Quantitative evaluation and quality control of total RNA was determined using a spectrophotometer—NanoDrop 2000 . Only RNA samples for which the absorbance ratio at wavelength 260nm/280nm was 1.8–2.1 were adopted for the next analysis steps. The mentioned absorbance ratio proves that isolated RNA is of high quality An aliquot of 1μg of total RNA was reverse transcribed into cDNA using iScript™ Advanced cDNA Synthesis Kit for RT-qPCR . cDNA synthesis was performed in a final volume of 20 μl using an Thermal Cycler . For reverse transcription, the mixtures were incubated at 46°C for 20 min then heated to 95°C for 1 min and finally rapidly cooled at 4°C.A heart sample . Specific primers for the β-catenin (CTNNB1), CacyBP/SIP (CACY BP), galectin-3 (LGALS3), LMP7 (PSMB8) and GAPDH were designed by BIO-RAD Company. The housekeeping gene GAPDH was used as the reference gene for quantification. In order to determine the amounts of tested genes expression levels, standard curves for each gene separately were constructed with serially diluted PCR products. PCR products were obtained by amplification of cDNA using specific primers as follows CTNNB1 , CACY BP , LGALS3 , PSMB8  and GAPDH . qRT-PCR was carried out in a dublete i.e. for each patient the expression of a gene in question was measured in two technical replicates. The qRT-PCR reaction was conducted in a final volume of 20 μl under the following conditions: 2 min polymerase activation at 95°C, 5 sec denaturation at 95°C, 30 sec annealing at 60°C for 35 cycles. PCR reactions were checked by including no-RT-controls, by omission of templates, and by melting curve to ensure only a single product was amplified. The relative quantification of gene expression was determined by comparison of values of Ct using the ΔΔCt method. All results were normalized to GAPDH.Quantitative real-time PCR reactions were performed using) Stratagene Mx3005P (Aligent Technologies) with the SsoAdvancedAll data were analysed for statistical significance using software computer package Statistica Version 12.0. The mean values were computed automatically; significant differences were determined by Student’s t-test; p<0.05 was accepted as significant. The relationships between age and intensity of immunostaining for β-catenin, CacyBP/SIP, galectin-3, LMP7 as well as correlation between age and expression of β-catenin, CacyBP/SIP, galectin-3, LMP7 genes were examined using the Pearson's correlation coefficient, p<0.05 was considered statistically significant.The results of the positive test with the antibodies used are shown in Mean values of age, body weight and body mass index (BMI) in the two studied groups of men are presented in During a routine histological assessment, no significant pathological changes were found in the hearts of the studied men. Positive immunohistochemistry with β-catenin, CacyBP/SIP, galectin-3 and LMP7 was observed in the hearts of all the men tested, although reaction intensity varied between different age groups Figs .Immunostaining of β-catenin in the hearts of men revealed its location in intercalated discs and in the perinuclear area of cardiomyocytes cytoplasm . In the CacyBP/SIP was identified in the man’s hearts in the form of small brown-stained granules located near the cardiomyocyte nuclei . CacyBP/Antibodies against galectin-3 gave a very weak or almost undetectable reaction in the myocardium of men under 45 , whereas2 section area) and increased width of cardiomyocytes (cardiomyocytes width 11.1 ± 0.28 μm) in the hearts of men over 45 years of age compared with younger men   .A test utilising qRT-PCR method showed an increase in the expression of β-catenin, CacyBP/SIP, galectin-3 and LMP7 genes in the heart of men over 45 years of age compared to men under 45 years of age , Table 4Ageing is one of the most significant risk factors for development of cardiovascular complications and mortality due to cardiovascular events , 2. AfteAs evident from the review of the literature, this is the first report on the comparative evaluation of the distribution of β-catenin, CacyBP/SIP, galectin-3 and LMP7 immunosubunit in the hearts of healthy men in different age groups. In the present study, we found a stronger immune response against β-catenin, CacyBP/SIP, galectin-3 and LMP7 in the hearts of men over 45 years of age compared to younger individuals.The present report also revealed increased, age-related, expression of the β-catenin, CacyBP/SIP, galectin-3 and LMP7 gene in the hearts of men over 45 years of age, which was consistent with immunohistochemical changes in peptide content.Our findings are consistent with experimental data showing increased levels of β-catenin in the heart and enhanced production of immunoproteasomes in skeletal muscles of ageing rodents , 35. SevAgeing is accompanied by histopathological changes in the heart, such as a successive loss of cardiomyocytes, myocardial hypertrophy and fibrotic remodelling of the heart , 39.In vitro studies by Hahn et al, [β-catenin, CacyBP/SIP and galectin-3 are crucial for the survival of cardiomyocytes. n et al,  and Bergn et al,  have revn et al,  demonstrn et al, . Exposurn et al, , 42. ConSome recent evidence underlines the profibrotic and hypertrophic effects of β-catenin and galectin-3 in the heart and shows on deleterious consequence of activating those peptides on cardiomyocytes. The modification of β-catenin gene, resulting in the expression of the non-degradable peptide form and its accumulation in the cytoplasm, provoked enlargement of isolated rat cardiomyocytes . SimilarIn our study, we observed significantly higher content of β-catenin in the intercalated discs in the heart of older men compared to those under the age 45 years. The presented results are consistent with a report by Bonda et al.  who founOxidative stress is considered to be one of the major pathomechanisms associated with the process of cell ageing and apoptosis . TopolskThe latest literature data indicate a significant impairment of proteasomes activity in the ageing heart , 29. It The results of quantitative and morphometric analyses revealed a decrease in the number of cardiomyocytes and the hypertrophy of cardiac muscle cells in men older than 45 years. In a study on the impact of aging on the human heart, Olivetti et al.  also obsOur research results clearly show that the aging process leads to disruption in the expression and content of β-catenin, CacyBP/SIP, galectin-3 and immunoproteasomes in heart cells.To the best of the authors' knowledge, this is the first study characterizing the hearts of healthy men for their expression of β-catenin, CacyBP/SIP, galectin-3 and immunoproteasome subunit LMP7. The differences in the expression of the examined parameters in the heart of men of different ages, demonstrated in the conducted studies, suggest participation of β-catenin, CacyBP/SIP, galectin-3 and LMP7 in the regulation of many important processes and heart homeostasis during ageing.S1 Fig(TIF)Click here for additional data file.


CNGA3 gene, which encodes the A subunit of the cyclic guanosine monophosphate (cGMP)-gated cation channel in cone photoreceptor outer segments, cause total colour blindness, also referred to as achromatopsia. Cones lacking this channel protein are non-functional, accumulate high levels of the second messenger cGMP and degenerate over time after induction of ER stress. The cell death mechanisms that lead to loss of affected cones are only partially understood. Here, we explored the disease mechanisms in the Cnga3 knockout (KO) mouse model of achromatopsia. We found that another important effector of cGMP, the cGMP-dependent protein kinase 2 (Prkg2) is crucially involved in cGMP cytotoxicity of cones in Cnga3 KO mice. Virus-mediated knockdown or genetic ablation of Prkg2 in Cnga3 KO mice counteracted degeneration and preserved the number of cones. Analysis of markers of endoplasmic reticulum stress and unfolded protein response confirmed that induction of these processes in Cnga3 KO cones also depends on Prkg2. In conclusion, we identified Prkg2 as a novel key mediator of cone photoreceptor degeneration in achromatopsia. Our data suggest that this cGMP mediator could be a novel pharmacological target for future neuroprotective therapies.Mutations in the  CNGA3 or CNGB3 [2+ signals [GNAT2, PDE6C, and PDE6H also encode proteins involved in the cone visual transduction cascade [ATF6, encoding an endoplasmic reticulum (ER)-localized transmembrane transcription factor that can activate the unfolded protein response (UPR) and plays a role in ER homeostasis [Achromatopsia (ACHM) is an inherited retinal disorder affecting retinal cones, the type of photoreceptors that mediate high acuity daylight vision. Cone outer segments, the specialized compartments of these photoreceptors, contain all proteins needed for light detection and conversion into chemical and electrical signals. Mutations in genes encoding key proteins of this cascade result in total colour blindness, also referred to as achromatopsia. Approximately 80 percent of ACHM patients carry mutations in one of the genes or CNGB3 , which eor CNGB3 . The con cascade . The sixeostasis .Like many other inherited disorders, ACHM manifests already in childhood with clinical symptoms that include lack of colour discrimination, poor visual acuity, extreme light sensitivity (photophobia), and involuntary eye movements (nystagmus) . Given tCNGA3- and CNGB3-linked ACHM [CNGA3-linked ACHM was already completed and recently reported promising safety and efficacy data [Currently, there is no treatment available for ACHM, but several groups are working on the development of gene supplementation therapies for both ked ACHM . One phaacy data . Three aThe concept of gene supplementation with AAV vectors is only applicable at early stages of the disease and providing a sufficient number of morphologically intact and, thus, rescuable cone photoreceptors is still present . Given tCnga3 knockout model of ACHM. We found that the membrane bound cGMP-dependent protein kinase 2 (Prkg2) is crucially involved in cGMP-mediated cytotoxicity in cones. Genetic depletion of Prkg2 resulted in a long-lasting preservation of cone photoreceptors. Mechanistically this neuroprotective effect of Prkg2 depletion on cones seems to involve inhibition of DNA damage, unfolded protein and ER-stress mechanisms. Our work highlights the cGMP kinase Prkg2 as a novel target for neuroprotective treatments aiming to preserve the morphology and structure of affected ACHM cone photoreceptors.Here, we investigated the role of cGMP signalling on viability of cone photoreceptors in the GUCY2E gene) and its levels are balanced by the cone phosphodiesterase  inhibits the retGC activity in a negative feedback mechanism. In cones missing the CNG channel, the lack in Ca2+ influx would likely determine the constant activation of retGC by Gcap1 and the production of excessive amounts of cGMP  promoter [Gucy2e downregulation  vector encoding a shRNA that targets the endogenous promoter . This trgulation c,d. Thiseceptors .Cnga3 KO cones, we explored the expression of the cGMP-dependent kinases, namely Prkg1 and Prkg2. As shown in Cnga3 KO retina at different postnatal time points, we observed a transient upregulation of Prkg2 transcript on week 2  as well as in Cnga3 KO and wildtype mice  functional magnetic microparticles (Ti-IMAC) to enrich phosphorylated peptides [Cnga3 KO, and Cnga3/Prkg2 DKO retinal protein lysates for subsequent label free quantification (LFQ) using mass spectrometry . Phosphorylated Atr peptides were found in Cnga3 KO retinal lysates, but were not detected in lysates from Cnga3/Prkg2 DKO or wildtype retina.Since Prkg2 is a cGMP-dependent serine/threonine-specific kinase, it is tempting to speculate that this neuroprotective effect is mediated by the lack of its kinase activity. We, therefore, designed an experiment to identify potential changes in the pattern of phosphoproteins in peptides  from wiltrometry . We reasThe MaxQuant analysis software  allows fCnga3 KO cone photoreceptors [Prkg2-deletion influences ER stress markers in Cnga3 KO cones. To facilitate the bulk analysis of cone photoreceptor markers, we generated a Cnga3/Prkg2 double-deficient mouse line on the cone-dominant Nrl KO background [Cnga3/Nrl DKO mice display a cone degeneration similar to the Cnga3 single KO [Cnga3/Nrl DKO and Cnga3/Nrl/Prkg2 triple knockout (TKO) mice. As shown in Prkg2 lead to a significant reduction of the Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) signal, indicative of a reduced cell death rate. We next analyzed the effect of Prkg2 knockout on the levels of phosphorylation of eukaryotic initiation factor 2 (p-eIF2a) which is an established ER stress marker already shown to be upregulated in Cnga3-deficient cone photoreceptors [Prkg2-deletion, the levels of p-eIF2a were reduced to a level similar to Cnga3/Nrl/Gucy2e TKO mice , and Ryr2, the gene encoding the type 2 ryanodine receptor (Ryr2). As depicted in Cnga3 KO context, which were normalized after additional knockout of Prkg2. A similar effect was observed for the unfolded protein response (UPR)-related genes Atf6b (activating transcription factor 6 beta), Bax (Bcl2-associated X protein), Cebpb (CCAAT/enhancer-binding protein beta), Creb3l3 (cAMP-responsive element-binding protein 3-like 3), Derl1 (degradation in ER protein 1), Dnajc3 , Ern2 , Ganc , Srebf2 (sterol regulatory element-binding factor 2), and Uggt2 , which affect rod and cone photoreceptors function. Until recently, there was no hope for patients suffering from blinding inherited retinal disorders. This has changed with the approval of a first gene therapy for a specific condition, RPE65-linked retinal dystrophy [Loss of vision is one of the most severe handicaps with high socioeconomic importance. Many blinding disorders are inherited and caused by mutation in one of more than 250 retinal disease genes  by phosphorylating downstream effector proteins [Cnga3 KO cones remains unclear. At this stage we can only speculate that cGMP-Prkg2 signalling triggers cell death by activating DNA damage pathways. This intriguing hypothesis deserves further investigation in the future, in particular since recent evidence suggests a cross-talk between DDR and ER stress [Using an approach based on Ti-IMAC enrichment of phosphorylated proteins in combination with proteins . Of noteproteins . Given tR stress .Prkg2 transcript at PW2, we can speculate that a transient upregulation of the cGMP-Prkg2 signalling around eye opening could trigger subsequent cell death processes. Since the progression of cone degeneration in the Cnga3 KO mouse is rather slow, we believe that multiple signaling processes running in parallel contribute to degeneration of cones; thus, Prkg2-dependent mechanisms are responsible only in part for the observed effects and do not dictate the kinetics of degeneration. Additionally, more focused experiments with Cnga3 KO mice on a cone-rich Nrl KO background are needed to clarify this issue. Further studies are also needed to get more insight into the timing of the pathophysiological events that eventually lead to cone cell death. From the upregulation of Taken together, this study establishes Prkg2 as a novel therapeutic target for neuroprotection of degenerating cone photoreceptors in achromatopsia, although the therapeutic applicability of Prkg2 inhibition needs to be further validated. Another aspect that remains to be elucidated is whether this neuroprotective effect is specific for cones or if it is also relevant for rod photoreceptors. Moreover, Prkg2 inhibition could provide protection not only from cGMP-mediated cytotoxicity, but also from degeneration caused by other detrimental stimuli. Importantly, in the case of inherited retinal disorders like achromatopsia, a genetic mutation causes a functional defect. Thus, the underlying gene defect needs to be corrected or a healthy copy of the affected gene needs to be supplemented in order to achieve an improvement in cone function. However, such gene correction or supplementation therapies are only applicable if a sufficient number of morphologically intact and, thus, rescuable target cells remains. Therefore, advanced stage patients who lost the majority of their photoreceptors are excluded from gene-specific gene therapies. Based on the presented results it is tempting to speculate that a neuroprotective therapy by Prkg2 inhibition has the potential to delay degeneration of cones and to widen the window of opportunity for gene therapy in achromatopsia.Cnga3/Prkg2 double knockout mice were generated by cross-breeding Cnga3 KO mice [Prkg2 KO mice [Cnga3/Nrl DKO and Cnga3/Nrl/Gucy2e TKO mice were generated by cross-breeding as described previously [Cnga3/Nrl/Prkg2 TKO mice were generated by cross-breeding Cnga3 KO mice with Prkg2 KO mice [Nrl KO [Cnga3 KO mice were also crossbred with RG-eGFP mice [Cnga3 KO mice. Mice were maintained on a 12 h light/dark cycle. During the light cycle, cage illumination was approximately 7 foot-candles.All animal experiments were performed according to the ARVO statement for the use of animals in ophthalmic and vision research and were approved by the local authorities . The project identification codes were ROB-55.2-2532.Vet_02-14-59 and 18-060-EAFI, respectively. All mice used were on a mixed C57-BL6/J-129/Sv genetic background. eviously ,35. Cnga [Nrl KO  mice. FoGFP mice  to generPrkg2 KO mouse, then cleared by centrifugation and used at a final dilution of 1:50 for immunohistochemistry. Confocal images were collected at a Leica TCS SP8 spectral confocal laser scanning microscope (Leica Microsystems). The generated files were processed with LAS X software and the open-source software Fiji [We prepared mouse eye cross-sections for immunohistochemical analysis as described previously ,38. Cellare Fiji ,40.The terminal deoxynucleotidyltransferase dUTP nick end-labelling (TUNEL) was performed and analysed as described previously ,35. ConfFor shRNA experiments a modified pSub201 plasmid  was geneCnga3 KO mice. Fundus fluorescence examination was performed with an adapted Spectralis HRA + OCT system from Heidelberg Engineering  in combination with optic lenses, as described previously [1 µL of titer-matched AAV8-shRNA vector containing approx. 5E10 total vector genomes was delivered via subretinal injection in 2-week-old RG-eGFP/eviously .Retinal protein preparation, SDS-PAGE, and western blot analysis were performed as described previously ,35. A LiTotal RNA preparation and reverse transcription were per- formed as described previously ,21. The g for 10 min, the supernatant was removed and the pellet was washed two times with 1 mL of 80% acetone each.After lysis, samples were incubated with 1 µL Benzonase for 1 h at 0 °C. Afterwards, 250 µL of MS-grade water was added and 2 retinas from the same date were pooled together. To precipitate the proteins, 7 mL of ice-cold acetone(aq) was added to each sample, followed by incubation at −20 °C overnight. After centrifugation at 10,000× To the resulting pellets, 2 mL of digestion-buffer  and 2 mL of MS-grade water were added and the samples were resuspended by sonication  on ice. Afterwards, the protein concentration of the samples was determined by conducting a Bradford assay.A volume equivalent of 445 µg protein content was taken. For digestion, samples were processed in the same way as for the full-proteome analysis with the following amounts: TCEP(aq) (1M) was added to a final concentration of 41.8 mM, 255 µL of TEAB(aq) (1M) were added to neutralize the solution, iodoacetamide(aq) (1M) was added to a final concentration of 41.1 mM and 210 µL of TEAB(aq) (1M) were added to adjust the pH-value for digestion. Finally, 5 µg trypsin was added per sample (3 µg in case of the IA-triplicate samples).After digestion, peptides were desalted by stage-tip purification  and con® magnetic Ti-IMAC beads  were prepared according to the manufacturer’s instructions. For loading, the dried peptides were dissolved in 100 µL loading-buffer  and incubated for 20 min at room temperature with end-over-top mixing with the prepared Ti-IMAC beads. Unbound sample was removed by washing two times with 100 µL loading buffer for 30 s. Subsequently, the beads were incubated four times with 100 µL wash buffer  for 2 min each. Phosphopeptides were eluted by incubation three times with 80 µL elution buffer (1% NH4OH in MS-grade water) for 15 min each.For each replicate, 50 µL MagReSynAfter elution, phosphopeptide-containing samples were acidified with FA(aq), desalted by stage-tip purification  and conThe phosphopeptide-samples were solved in 12 µL MS-solvent.The samples were analyzed with an UltiMate 3000 RSLCnano liquid chromatography system  attached to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). They were concentrated on a µ-precolumn cartridge ) and further processed on an in house packed analytical column , packed into a 75 µm i.d. × 150 mm fused silica picotip emitter with a 8 µm tip .The samples were processed via a 120 min multi-step analytical separation at a flow rate of 300 nL/min and a column temperature of 30 °C. Only LC-MS grade solvents were used . The gradient with percentages of solvent B was programmed in the following way: 1% for 3 min; from 1% to 6% in 2 min; from 6% to 34% in 85 min; from 34% to 60% in 10 min; from 60% to 85% in 5 min; 85% for 7 min; from 85% to 1% in 3 min; 1% for 5 min.Per analysis, 11 µL of the full proteome samples and 11.5 µL of the phosphopeptide samples were injected.Mass spectrometric analysis was done with a full mass scan in the mass range between m/z 300 and 1750 at a resolution of 120,000, an AGC target of 3e6 charges and a maximum ion injection time of 20 ms in profile mode. Following this survey scan, the 15 most intense ions were selected, fragmented and measured in profile mode with the following parameters: resolution of 15,000; AGC target of 2e5 charges; maximum ion injection time of 100 ms; isolation window of 2 m/z, with an offset of +0.3 m/z; stepped normalized HCD energy of 20%, 25% and 30%. Signals with an unrecognized charge state or a charge state of 1, 7, 8 or higher were not picked for fragmentation. To avoid supersampling of the peptides, signals were excluded from the analysis for 25 s after being selected for isolation and fragmentation. The peptide match setting was set to “-” and the exclude isotope setting was set to “on”.Data analysis of the phosphor-enriched samples was performed with MaxQuant version 1.5.1.0  with the following settings: variable modifications of acetyl (protein N-term), oxidation (M) and phosphorylation (STY); fixed modification of carbamidomethyl on cysteines. FDR-levels were set to 0.01. Fast LFQ was performed for quantification. The “match between runs”-feature was enabled. A mus musculus FASTA (proteome-ID: UP000000589) was used from uniprot. Trypsin was set as the protease and a maximum of 2 missed cleavages and a minimal peptide length of seven was chosen. Further data analysis was performed with Perseus. To do so, LFQ intensities obtained from MaxQuant analysis were log2-transformed and only proteins identified in at least three of the retinal tissue samples were retained.p < 0.05. Data were analysed and graphed using the GraphPad Prism software (GraphPad Software).One-way analysis of variance and unpaired Student’s t test were used to evaluate significant differences between multiple groups and two groups, respectively. Differences were considered statistically significant when


Sacrococcygeal teratoma is rare and happens in 1:35,000 to 40,000 live births. It is more common in girls than boys with the reported ratio of 3:1 to 4:1. We herein report an unusual case of a huge sacrococcygeal teratoma, which was more than half of the size and weight of the baby which was terminated at 24 weeks of gestation. The tumors have multiple tissue types as it is composed of two or three germ cell layers. Though SCT are more prevalent in neonates, infants and children, it has been reported in adult population also. Mostly, sacrococcygeal tumours are cystic and benign whereas malignant tumours comprises 1-2%.4 Due to high vascularity in some solid tumours, it can cause problem during pregnancy and the postoperative period. Prenatal and perinatal complications may occur more commonly in fetus with sacrococcygeal teratoma. The natural history and pathophysiology of fetal SCT is different than that of postnatally diagnosed SCT. With optimal obstetric and surgical management as well as anticipation and recognition of pathophysiologic events, fetuses can be managed to survive. In a subset of fetuses with SCT, fetal resection of tumor may offer the only hope for survival.Sacrococcygeal teratoma, which is mostly present in infancy, is a tumour located at the base of coccyx. It is thought to be derived from embryonic germ cell layers.A 19 years old primigravida at 24 weeks gestation was referred for anomaly scan in our center. She had her regular ANC visit in another hospital outside Kathmandu. In anomaly scan , a large space-occupying lesion measuring 123 × 107 × 126 mm(Volume = 870cc) in right side of fetus with high vascularity, and solid component seen to be attached to posterior side of fetus near by sacrococygeal area . She hadHistopathology report of the specimen confirmed immature sacrococcygeal teratoma of grade three with lymphovascular and peri-neural invasions not identified. .5 They are believed to arise from the remnant of the primitive streak in the coccygeal region during the early third week of gestation from the totipotential cells of Hensen's node. The first case of sacrococcygeal teratoma has been reported on a Chaldean cuneiform tablet dated approximately 2000 BC.6 According to Keslar et al reports, 69 (62%) of the 96 sacrococcygeal teratomas in their series were composed of both solid and cystic elements.7 The cystic type may be filled with various components such as serous fluid, mucoid, or sebaceous material and are lined by true epithelium. The most common site for sacrococcygeal teratoma is coccygeal region followed by less common sites such as mediastinum, testes, retroperitoneum, brain, head and neck, vagina, stomach, and pineal region.7 They present mostly during the gestation period of between the 22nd and the 34th week of gestation. Sacrococcygeal teratoma when diagnosed on routine sonograms is linked with complications related to perinatal and preenatal period.8Tissues of Sacrococcygeal teratomas arise from all the germ layers ectoderm, mesoderm, and endoderm.10 However, tumour markers are not seen in the benign type.Depending on the location and extension, clinical manifestations of the SCT may present with low back pain, bowel or urinary symptoms and venous engorgement of the lower limbs which may be related to the mass effects of the tumour. In malignant lesions, markers of biochemical origin such as AFP, carcinoembryonic antigen and HCG are elevated and help to detect recurrences after operation.11 SCT, being large enough in size, exerts a significant mass effect on adjacent structures during fetal period and results in various co-morbidities such as obstructive hydronephrosis, anterior displacement of the anorectum, bowel distension, hypoplasia of pelvic muscles, mucocolpos or hip dislocation.12Fetal hydrops can occur in the tumour due to high output cardiac failure and anemia which results from arterio-venous shunting and intramural hemorrhage. That is why, fetal hydrops is considered as a prognostic factor for fetuses with SCT and warrants mandatory prenatal careful monitoring.13 Therefore, in order to overcome the limitations of the ultrasound, CT and MRI are now used for preoperative examination so as to determine the structure, vascularity, exact location, and components of a tumor and its relationship to the surrounding structures.15 The main treatment for sacrococcygeal teratoma is the complete surgical resection. This can be done by either anterior approach (for tumours extending above the S3 region) or posterior approach (for tumours below S3 region).17 For large tumours which can not be excised completely can be operated by a combined anteriorposterior approach.18 Pre-operative CT and MRI helps in choosing the appropriate path for operative procedure of an SCT.Although ultrasonography has been the imaging modality of choice during pregnancy as it is a safe method, cost-efficient and widely available, it has few limitations such as limited field of view and difficulties in penetrating bones.JNMA Case Report Consent Form was signedby the patient and the original article is attached withthe patient's chart.None.


Gastric cancer (GC) has a high mortality rate, and metastasis is the main reason for treatment failure. It is important to study the mechanism of tumour invasion and metastasis based on the regulation of key genes. In a previous study comparing the expression differences between GES-1 and SGC-7901 cells, PCDHGA9 was selected for further research. In vitro and in vivo experiments showed that PCDHGA9 inhibited invasion and metastasis. A cluster analysis suggested that PCDHGA9 inhibited epithelial-mesenchymal transition (EMT) through the Wnt/β-catenin and TGF-β pathways. Laser confocal techniques and western blotting revealed that PCDHGA9 inhibited the nuclear translocation of β-catenin, regulated T cell factor (TCF)/ /lymphoid enhancer factor (LEF) transcriptional activity, directly impacted the signal transmission of the TGF-β/Smad2/3 pathway, strengthened the adhesion complex, weakened the effects of TGF-β, and blocked the activation of the Wnt pathway. In addition, PCDHGA9 expression was regulated by methylation, which was closely related to poor clinical prognosis. The aim of this study was to elucidate the molecular mechanism by which PCDHGA9 inhibits EMT and metastasis in GC to provide a new theoretical basis for identifying GC metastasis and a new target for improving the outcome of metastatic GC. With its poor prognosis, overall 5-year survival rate of GC is still less than 30%, and distant metastasis is the major barrier to improve the therapeutic effect3. Tumour metastasis is a multistep and multi-molecular process4; therefore, a thorough understanding of the mechanism underlying GC metastasis is significant for developing innovative therapeutic tactics.Gastric cancer (GC) was the second leading cause of cancer-related death and the sixth most frequently diagnosed cancer worldwide in 20185. Importantly, epithelial cells undergo a developmental switch that enables them to acquire mesenchymal characteristics, resulting in a decrease in adhesion and cell polarity and an increase in motility and invasiveness during EMT6. This process is also associated with upregulation of N-cadherin, Vimentin and Slug and concomitant downregulation of E-cadherin7. EMT involves complex mechanisms regulated by many signalling pathways, including the Wnt/β-catenin pathway, the transforming growth factor-β (TGF-β)/Smad2/3 pathway and other pathways9. Accumulating evidence indicates that the canonical Wnt pathway negatively regulates E-cadherin and induces EMT by protecting the significant factor β-catenin from proteasome degradation11. Normally, β-catenin interacts with cadherin and forms a complex at the membrane. TGF-β may disassociate this complex to release β-catenin, which can subsequently translocate to the nucleus; this is required for posttranscriptional regulation of β-catenin and activation of EMT12.Epithelial-mesenchymal transition (EMT) is essential in the initial stages of GC metastasis; in this process, the epithelial cell cytoskeleton is reorganized, and tight junctions are dissolved14. As members of the cadherin family, protocadherins (PCDHs) likely play critical roles in the establishment and function of specific cell–cell connections in the brain15. However, little information is available about the relationship between PCDHs and either tumorigenesis or nuclear signals. Our previous study indicates that PCDHGA9 may serve as a potential novel biomarker in GC and is closely associated with GC patient outcomes16. Nevertheless, we have not determined how PCDHGA9 is downregulated in GC. It is well known that the occurrence and development of GC are characterized by the gradual formation of multiple epigenetic and genetic mutations. DNA methylation could cause promoter hypermethylation and specific gene inactivation19. Here, we assessed the methylation and inactivation frequency of PCDHGA9 in cancer tissues and investigated its functions in the progression of GC. In our previous study, we clearly demonstrated that PCDHGA9 suppresses GC cell proliferation via the Wnt/β-catenin pathway and inhibits EMT by suppressing TGF-β/Smad2/3 pathway activation. Importantly, we analysed cDNA array information via Ingenuity Pathway Analysis (IPA) and found that there might be a connection between the Wnt/β-catenin and TGF-β/Smad2/3 pathways in EMT signalling. In the present study, we further determined that PCDHGA9 could directly interact with β-catenin to form a complex at the GC cell membrane to inhibit EMT, and we provide evidence of the association between the canonical Wnt pathway and the TGF-β pathway.According to some models, downregulation of cadherin leads to a reduction in β-catenin membrane binding, mediating its effect on gene transcriptionIn this study, we demonstrated that PCDHGA9 is downregulated in GC tissues, especially in metastatic GC. Moreover, we found that the loss of PCDHGA9 results in the nuclear translocation of β-catenin and the promotion of EMT in GC cells, leading to enhanced metastatic and invasive abilities. Furthermore, we revealed a negative correlation between PCDHGA9 and N-cadherin, Vimentin and Twist and a positive correlation between PCDHGA9 and E-cadherin expression in GC specimens. In addition, we showed that PCDHGA9 interacts with β-catenin to antagonize the canonical Wnt pathway and inhibit the TGF-β/Smad2/3 pathway. Importantly, promoter hypermethylation was correlated with PCDHGA9 downregulation and poor prognosis in GC patients. Taken together, our data show that PCDHGA9 is a novel Wnt/β-catenin signalling pathway suppressor and a potential therapeutic target in metastatic GC.A total of 66 pairs of fresh frozen GC and normal tissue specimens were obtained in accordance with the guidelines approved by the institutional review boards of Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine. None of the patients received chemotherapy, radiotherapy or other related antitumour therapies before surgery. The final patient follow-up date was 1 August 2009, and the median observation time for the patients was 37.5 months . At least two certified pathologists confirmed all diagnoses, and the tumour stage and grade classification were based on pathological findings according to International Union Against Cancer guidelines. This study was conducted in accordance with the World Medical Association Declaration of Helsinki. All patients signed informed consent forms. The clinicopathological characteristics of the patients are summarized in Table −(ΔCt sample–△ΔCt control) method, and all experiments were run in triplicate.Total RNA from cell lines and matched primary and normal mucosa tissues from GC patients was extracted using TRIzol reagent . cDNA was reverse transcribed from 2 μg of total RNA using a HiScript qRT SuperMix for qPCR kit  according to the manufacturer’s instructions. qRT-PCR was performed using a ChamQ SYBR qPCR Master Mix kit . The primers used are listed in Table Total protein or nuclear protein was extracted according to the manufacturer’s instructions . Protein concentrations were measured using a BCA assay . Equal amounts of protein (50 μg) were separated on SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes . The membranes were first blocked for nonspecific binding in 5% skim milk in TBST buffer at room temperature for 1.5 h, incubated with specific primary antibodies at 4 °C overnight, and incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. The bound antibody was detected with ECL reagent . The primary antibodies for western blotting are listed in Table The human GC cell lines SGC-7901, MGC-803, MKN-45, MKN-28, HGC-27, AGS, BGC-823 and GES-1 were obtained from the Type Culture Collection of the Chinese Academy of Sciences . The bisulfite sequencing PCR (BSP) primers were as follows: PCDHGA9-1, forward 5′-GTTATGGGAGGTTAGGGTTAG-3′ and reverse 5′-AATCAATAACTTCTAAAAATAAATAAC-3′; and PCDHGA9-2, forward 5′-GTTATTTATTTTTAGAAGTTATTGATT-3′ and reverse 5′-CAAAATTTATATACCACTTTTCC-3′. The pLVX-IRES-puro-3×Flag-hPCDHGA9 and pHis-CMV4-hβ-catenin plasmids were used to ectopically express PCDHGA9 and β-catenin, respectively. The shRNA target sequences were as follows: sh-PCDHGA9-1, GCGGAAGATTAGTCCTGCTAT; sh-PCDHGA9-2, CCCAAATTCTTGACCGAGAAA and sh-PCDHGA9-3, CCTGCAAGTGACTGACATCAA.t-test was used to analyse the time-course data.MKN-28, MKN-45, SGC-7901 and MGC-803 cells with stable plasmid transfection were seeded in 6-well plates. After the cells reached confluence, a linear uniform scratch was generated with a standard 10-μl pipette tip at the centre of the well. Photographs were captured at the indicated time intervals in the different cell lines, and wound widths were quantified and compared to baseline values in experiments that were repeated three times. A two-tailed unpaired 4/chamber) was seeded in 200 μl of serum-free media in the upper chamber of an 8.0-μm pore transwell  without (migration) or with (invasion) Matrigel . The lower chamber contained 600 μl of medium supplemented with 10% FBS as a chemokine. After 24 h of incubation, the cells that migrated or invaded to the membrane surface exposed to the lower chamber were fixed with methyl alcohol, stained with a 0.1% crystal violet solution and then counted in 10 random fields. The experiments were repeated in triplicate.Each group of cells (2 × 106 cells were injected subcutaneously into the groins of nude mice (n = 6 for each group). Tumour diameter was measured every 5 days. One month later, the mice were sacrificed, and tumours were harvested. The tumour volumes were calculated using the formula (volume = length × width2 × 1/2).For the subcutaneous tumour growth assay, 150 μl of 2 × 106 stable cells were injected into the abdominal cavities of nude mice (n = 6 per group). After 1 month, all the mice were sacrificed, and the number of peritoneal metastatic nodules was counted. Fresh subcutaneous tumour and metastatic nodule tissues were removed and stored at −80 °C.For the peritoneal metastatic model, 1 × 106 cells stably transduced with plasmid were injected into the spleen. Then, the abdomen was closed (n = 6 per group). Aseptic techniques were strictly followed during the entire operation. Six weeks later, all the mice were sacrificed, and the livers were harvested and fixed with 4% paraformaldehyde for H&E staining. Then, the number of metastatic nodules was counted.For the hepatic metastasis assay, mice were anaesthetized, and a lateral incision was made in the abdomen. Fifty microliters containing 1 × 10Animal experiments were conducted with 4-week-old male BALB/c nude mice . All animal assays were approved by the Institutional Animal Care and Use Committee of Shanghai General Hospital.Cells were seeded onto sterilized coverslips, incubated for 48 h, washed once with PBS, fixed in 4% paraformaldehyde for 20 min at room temperature, washed three times with PBS and permeabilized with 0.1% Triton X-100 for 3 min. Then, the cells were washed three times with PBS and blocked for 1 h with blocking buffer .The coverslips were incubated overnight with an antibody against β-catenin (1:100), followed by an additional 2 h incubation with a fluorescently labelled secondary antibody  in PBS. Cell nuclei were stained with 4′,6 diamidino-2-phenylindole (DAPI) for 1 min, and the slides were viewed under a confocal laser-scanning microscope .Total protein from 293 T cells was extracted using RIPA lysis buffer. Cell lysates were immunoprecipitated with the indicated antibodies conjugated to protein A/G beads (Santa Cruz Biotechnology) overnight. Then, the beads were washed and boiled in SDS loading buffer. Using western blotting, the immunoprecipitated protein complexes were detected.4 cells per well. After 24 h, the TOP flash or negative control FOP reporter plasmid  and a Renilla luciferase plasmid were introduced into these cells. Twenty-four hours later, the luciferase activity was measured with a dual-luciferase reporter kit .The indicated cells were seeded in 24-well plates at 2 × 10XAV-939 and CHIR-99021 were obtained from MedChemExpress . TGF-β1 was purchased from Peprotech . 5-Aza was obtained from Sigma-Aldrich .2 test. The correlations between PCDHGA9 and E-cadherin, N-cadherin, Vimentin or Twist were determined using the Spearman rank correlation. Metastasis-free survival (MFS) in patients was analysed by the Kaplan-Meier method with a log-rank test. All statistical analyses were carried out using the SPSS 22.0 statistical software package . A p-value <0.05 indicated a significant difference.Data were analyzed using one-way analysis of variance or Student’s t-test for comparison between groups. The associations between PCDHGA9 expression and other clinicopathological characteristics were analysed by χA cDNA microarray was used to assess differentially expressed genes between the normal gastric mucosa cell line GES-1 and the highly invasive GC cell line SGC-7901 Fig. . We founIn this study, we examined PCDHGA9 expression in GC cell lines and 66 matched pairs of fresh frozen GC and normal adjacent tissues with qRT-PCR. The results showed that PCDHGA9 expression was significantly reduced in GC cell lines and GC tissues Fig. . We thenp < 0.001) but negative associations between PCDHGA9 and N-cadherin , Vimentin  and Twist  expression. Taken together, these results suggest that PCDHGA9 is not only correlated with the clinicopathological characteristics of GC but also involved in EMT.Next, we examined the expression levels of EMT-related markers  and their association with PCDHGA9 expression in the above 66 GC specimens via qRT-PCR. Low PCDHGA9 expression was frequently correlated with low E-cadherin expression and high N-cadherin, Vimentin and Twist expression in tumour tissues Fig. . PearsonTo explore the effect of PCDHGA9 on EMT in GC cells, we assessed E-cadherin and Vimentin expression in different GC cell lines Sup Fig. . MKN-28 Next, we investigated the metastatic ability of GC cells with different expression levels of PCDHGA9. The wound healing assays suggested that cells with higher PCDHGA9 expression demonstrated significantly slower wound closure than their respective control cells Fig. . These rWe previously found that PCDHGA9 expression was associated with EMT-related markers in clinical GC specimens. Thus, we explored whether EMT was involved in the PCDHGA9-mediated alterations in cell motility. Upregulation of PCDHGA9 reduced Vimentin, Twist, Slug and N-cadherin expression and increased E-cadherin expression in SGC-7901 and MGC-803 cells at both the mRNA and protein levels Fig. . ConversNegative control cells and cells with stable PCDHGA9 overexpression or knockdown were subcutaneously inoculated into nude mice, and tumour growth was measured every 5 days after implantation. Cells with lower PCDHGA9 expression had significantly larger tumour sizes and faster xenograft formation than did the respective control cells Fig. . We nextIn addition, we assessed the effect of PCDHGA9 expression on liver metastasis by injecting modified MKN-28 and SGC-7901 cells into the spleens of nude mice. All mice in the MKN-28-KD1 group (6/6) showed liver metastasis, but fewer mice in the control group had liver metastasis (2/6). Furthermore, PCDHGA9 upregulation in SGC-7901 cells reduced the number of liver metastatic nodules in mice. Representative MRI and HE staining images of the livers from different groups are shown in Fig. Our previous study presented the hypothesis that PCDHGA9, a membrane protein that interacts with β-catenin, constitutes cell–cell junctions and prevents the dissociation of β-catenin from the membrane and its destruction by the degradation complex. To verify this hypothesis, we blocked or upregulated PCDHGA9 expression in different cells and observed the cellular distribution of β-catenin. After cells were cultured, total protein was extracted to create whole-cell lysate, membrane fractions and nuclear fractions, which were then assessed for β-catenin expression using western blotting. The results suggested that PCDHGA9 increased the membrane localization and decreased the nuclear translocation of β-catenin Fig. . Second,20. To further investigate whether PCDHGA9 exerts its effects during GC metastasis in the context of the Wnt/β-catenin pathway, we introduced β-catenin into the PCDHGA9-silenced MKN-28 and PCDHGA9-overexpressing SGC-7901 stable cell lines. As shown in Fig. β-Catenin plays a significant role in EMT and cancer metastasis and is the central protein of the Wnt signalling pathwayThe TOP/FOP ratio was increased when CHIR-99021, a specific activator of Wnt/β-catenin signalling, was employed, whereas PCDHGA9 overexpression weakened this effect Fig. . By cont21. TGF-β activates EMT by disassociating such complexes to release β-catenin22. The Wnt/β-catenin pathway protects dissociated β-catenin from degradation in the cytoplasm23. Nevertheless, the cadherin/β-catenin complex provides a powerful force for cell–cell adhesion24.Under homeostatic conditions, most β-catenin and cadherin molecules are localized to the membrane as an adhesion complexTo explore the relationships among PCDHGA9, TGF-β and Wnt/β-catenin, we investigated whether PCDHGA9 can interact with β-catenin, reduce the effect of TGF-β, block activation of the Wnt/β-catenin pathway and attenuate EMT. SGC-7901 cells were treated with TGF-β1 for 48 h, thus activating EMT, as evidenced by increased nuclear β-catenin levels, upregulation of Vimentin and N-cadherin expression, and downregulation of E-cadherin expression. However, using XAV-939 to inhibit the Wnt pathway significantly weakened the effects caused by TGF-β1 Fig. . FurtherWe found that PCDHGA9 is readily expressed in normal digestive tissues, especially in the stomach Fig. . As mentTo further explore the clinical association between the expression and methylation of PCDHGA9 and GC metastasis, we analysed the MFS of 66 GC patients. These patients were divided into two groups based on the median expression of PCDHGA9, and GC patients with high PCDHGA9 expression had a better prognosis and MFS Fig. . We alsoIn this study, we proposed that the membrane protein PCDHGA9 represses the delivery of nuclear signals and suppresses the progression of GC. The results demonstrate that PCDHGA9 interacts directly with β-catenin and reduces the dissociation of β-catenin from the membrane to antagonize the Wnt/β-catenin pathway and inhibit EMT. Moreover, we found that PCDHGA9 is downregulated in GC tissues and GC cell lines and that this downregulation is accompanied by hypermethylation at the PCDHGA9 promoter.On the basis of the cDNA array results, we identified candidate genes, from which PCDHGA9 was shown to significantly suppress GC cell migration and upregulate N-cadherin and Vimentin expression more than the other genes. The IHC results from the clinical samples revealed that PCDHGA9 expression was lower in GC tissues than in matched normal mucosa samples, and patients with liver metastasis showed significantly lower PCDHGA9 expression than did patients without metastasis. The in vitro assays demonstrated that PCDHGA9 played a negative role in regulating the migration and invasion properties of GC cells. Furthermore, we showed the suppressive effects of PCDHGA9 on the multiple stages of metastatic GC in vivo by employing subcutaneous implantation, peritoneal injection and liver metastasis models.10. Numerous studies support the notion that EMT activation induces the sequential cascade of metastasis25. Thus, exploring the key factors affecting this biological change is becoming increasingly significant. PCDHGA9 is a member of the cadherin family that is expressed diffusely in normal brain, spleen, colon and gastric tissues but is weakly expressed or silent in GC tissues. Our previous study indicated that PCDHGA9 could induce GC cell autophagy, apoptosis and cell cycle arrest and inhibit EMT via the TGF-β/Smad2/3 pathway. To further explore the relationship between PCDHGA9 and EMT, we evaluated PCDHGA9 expression and the molecular hallmarks of EMT and found a positive correlation between PCDHGA9 and E-cadherin expression but a negative correlation between PCDHGA9 and N-cadherin, Vimentin and Twist expression in clinical GC tissues, which provided convincing evidence that PCDHGA9 inhibits EMT in GC metastasis. Furthermore, PCDHGA9 overexpression in GC cells enhanced E-cadherin expression and reduced N-cadherin, Vimentin, Slug and Twist expression, whereas PCDHGA9 knockdown elicited opposing effects. We also observed similar results in animal models by extracting RNA from xenograft tumours and peritoneal metastatic nodules. Taken together, these findings suggest the potential effects of PCDHGA9 on EMT, which might be responsible for the metastatic and invasive potential of these cells.Tumour invasion and metastasis are complex and multistage processes in which tumour cells lose their cellular junctions and acquire characteristics that permit them to migrate through the matrix layer and invade into the circulation26. Recent research indicates that cadherin downregulation leads to the disruption of cell–cell junctions, which is the crucial step in EMT that contributes to GC metastasis27. Specifically, abnormal β-catenin distribution, which is indicative of Wnt/β-catenin pathway activation, is widely observed in GC28. β-Catenin translocation from the membrane to the nucleus initiates TCF/LEF transcription29, resulting in increases in Snail and Twist expression and decreases in E-cadherin expression, both of which are significantly important for EMT in GC progression24. PCDHGA9 is a member of the cadherin family, but its effect on nuclear signal delivery and EMT in GC cells remains unclear. Interestingly, upon exploring the subcellular localization of β-catenin by western blotting and confocal microscopy, we observed increased membrane β-catenin levels and decreased nuclear β-catenin levels in PCDHGA9-overexpressing GC cells, whereas PCDHGA9-silenced cells showed the opposite phenomenon, which may indicate that PCDHGA9 affects β-catenin distribution and strengthens the stability of membrane-bound β-catenin. Using immunoprecipitation and immunoblotting, we further confirmed that PCDHGA9 directly interacted with β-catenin at exogenous levels. The TOP/FOP luciferase activity assay showed that the transcriptional activity of β-catenin/TCF was inhibited by PCDHGA9 overexpression and that the target proteins of the Wnt/β-catenin pathway were suppressed upon ectopic overexpression of PCDHGA9. In addition, we found that ectopic expression of β-catenin upregulated proteins downstream of the Wnt/β-catenin pathway and induced EMT in GC cells, which could be reversed by PCDHGA9 overexpression. We also detected that TCF transcription activation and the changes in both EMT marker expression and GC cell invasion and migration induced by PCDHGA9 overexpression or silencing were reversed by either CHIR-99021 or XAV-939. Therefore, we conclude that PCDHGA9 can block the canonical Wnt pathway to inhibit EMT by interacting with β-catenin and reducing its dissociation.In most epithelial tissues, one common connection among cells is the adhesion complex, which is composed of cadherin and β-catenin9; this finding is in line with our previous hypothesis based on the IPA results that there exists some connections between the Wnt/β-catenin and TGF-β signalling pathways in EMT. We elaborated that in response to TGF-β signals from the microenvironment, the cadherin/β-catenin complex disassociates to release β-catenin30, and PCDHGA9 overexpression could reduce this effect and consequently suppress EMT in GC. In the present study, after using TGF-β to induce EMT, we observed that the nuclear translocation of β-catenin or induction of EMT could be reversed by XAV-939. Likewise, enhanced PCDHGA9 expression was also capable of reducing the effects of TGF-β. Thus, we conclude that PCDHGA9 suppresses GC metastasis and attenuates EMT induced by TGF-β and Wnt/β-catenin pathway activation. In this study, we found that PCDHGA9 was abundantly expressed in normal gastric mucosae and other digestive organs but silenced in most GC tissues and GC cell lines in accordance with hypermethylation of its promoter. Importantly, treatment with 5-Aza, a DNA methylation inhibitor, restored PCDHGA9 expression in the GC cell line, which indicated that downregulation of PCDHGA9 was partly mediated by DNA promoter methylation. Furthermore, we explored the clinical significance of PCDHGA9 by analysing the clinicopathologic features of 66 GC patients and found that patients with advanced GC had weak PCDHGA9 expression. Low PCDHGA9 expression (p = 0.0162) and PCDHGA9 promoter hypermethylation (p = 0.0469) were significantly correlated with MFS. Interestingly, compared with either element alone, the combination of PCDHGA9 expression and promoter hypermethylation reached a higher statistical significance in estimating MFS (p = 0.004), which may provide individual GC patients with a more precise appraisal of metastatic risk.Gonzalez DM reported the crosstalk of several signalling pathways closely related to EMTIn summary, this study illuminates the significance of PCDHGA9 in the suppression of GC metastasis through a β-catenin-dependent mechanism. PCDHGA9 interacts with β-catenin directly and weakens the effects of TGF-β and the Wnt/β-catenin pathway in the induction of EMT. Promoter hypermethylation and loss of PCDHGA9 expression in GC may indicate an aggressive phenotype and correlate with poor clinical survival. Moreover, demethylation of PCDHGA9 could be an innovative therapeutic target for GC invasion and metastasis. Therefore, the regulatory mechanism of PCDHGA9 expression and specific reagents that modify the methylation status of PCDHGA9 still require our attention.Supplementary Figure LegendsSupplematery Figure 1Supplematery Figure 2Supplematery Figure 3Supplematery Figure 4


P < .05). Finally, we confirmed Wnt/β‐catenin signalling achieves bile duct regeneration by regulating the expression of Lgr5 genes in DRCs (P < .05). We described the regenerative potential of DRCs and reveal opportunities and source for the treatment of cholestatic liver diseases.Recently, the incidence of bile duct‐related diseases continues to increase, and there is no effective drug treatment except liver transplantation. However, due to the limited liver source and expensive donations, clinical application is often limited. Although current studies have shown that ductular reaction cells (DRCs) reside in the vicinity of peribiliary glands can differentiate into cholangiocytes and would be an effective alternative to liver transplantation, the role and mechanism of DRCs in cholangiole physiology and bile duct injury remain unclear. A 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC)‐enriched diet was used to stimulate DRCs proliferation. Our research suggests DRCs are a type of intermediate stem cells with proliferative potential that exist in the bile duct injury. Meanwhile, DRCs have bidirectional differentiation potential, which can differentiate into hepatocytes and cholangiocytes. Furthermore, we found DRCs highly express Lgr5, and Lgr5 is a molecular marker for neonatal DRCs ( The bile duct system is divided into intrahepatic and extrahepatic bile ducts, the intrahepatic biliary system acting as a liver bracket plays an important role in the process of liver repair, and the proliferation of bile duct cells directly regulates the radius of the bile duct.Biliary epithelial cells in extrahepatic large bile duct and intrahepatic small bile duct are not homogeneous, showing obvious heterogeneity in morphology, biochemistry and function.Ductular reaction (DR) is a common injury response in lots of hepatobiliary diseases and is characterized by the proliferation of DRCs.In the previous work, we were surprised to find a class of intermediate cells resemble progenitor cell in DDC induced bile duct injury mice model, which were defined as abnormal hyperplasia of DRCs. These cells can differentiate into hepatocytes and bile duct cells, which are most likely important precursors of nascent bile duct cells. We thought it will be a new way for future treatment of biliary tract‐related diseases to clarify the source of new cholangiocytes and carry out cell transplantation technology.We separated DRCs from mice fed with DDC‐enriched diet, a robust model of bile duct injury and biliary fibrosis, suggesting a good platform for isolating DRCs.In DDC mice model, we found DRCs proliferate at the capillary bile duct and move radially from the portal area to the hepatic parenchymal region. Some DRCs further differentiate into mature bile duct cells. This process is an important stage to achieve structural reconstruction of damaged bile duct tree and repair of liver tissue function. It can be seen that the effective coordination between pathological amplification and normal maturation of DRCs plays a crucial role in bile duct cells regeneration, and the study of its regulatory mechanism has also become a hot topic, also, it is the key to the treatment of bile duct injury‐related diseases.β‐catenin signalling, although its ligand remains elusive, it has been shown that costimulation with Rspos 1 and Wnt‐3a induces increased internalization of Lgr5.β‐catenin‐dependent Wnt signalling.β‐catenin, which acts as a gene transcription activator by combining with nuclear complex T‐cell factor/lymphoid enhancer factor (TCF/LEF)..β‐catenin accumulates in the cytoplasm and transfers to the nucleus to initiate transcription of the target gene.We were more surprised to find Leucine‐rich repeat‐containing G protein‐coupled receptor 5 (Lgr5), a stem cell marker that is rarely expressed in general, showed enhanced expression in neonatal DRCs. Lgr5 also known as G protein‐coupled receptor 49/67 (Gpr49 or Gpr67) is a protein that in humans is encoded by the Lgr5 gene.β‐catenin with Apc deletion in mouse hepatocytes stably induced premature differentiation into BECs.β‐catenin signal to play an active role in biliary tract induction. However, so far, the role of Wnt signalling in the repair and regeneration of bile duct injury is still unclear. Therefore, we speculated Lgr5 may be a specific marker for such intermediate cells, and the differentiation process of DRCs is regulated by Wnt signalling pathway.Increasing evidence supports the important role of β‐catenin in regulating hepatocyte proliferation during development and regeneration. In the study of liver culture in vitro, Wnt‐3a promoted the differentiation of hepatocytes into biliary epithelial cells (BECs),In this study, we applied a Lgr5 CreERT2 DDC mouse model that enables us to identify and study DRCs. We have utilized various anatomic and immunohistochemical features including CK19 and CK7 as surface markers of cholangiocyte development,β‐catenin signalling pathway and its regulatory mechanism involved in intrahepatic biliary regeneration through DRCs proliferation, but also provides novel therapeutic targets in hepatobiliary disease for further study.This study not only extends our understanding of the Wnt/22.1The Lgr5‐CreERT2  mouse strain was purchased from the Model Animal Research Center of Nanjing University. All mice were kept under barrier conditions and were raised in our laboratory. After all mice were adapted to normal diet and drinking for one week, set up normal diet control group and DDC diet experimental group. The DDC model mice were fed with 1% DDC (Sigma‐Aldrich) for 4 weeks and it mainly caused bile duct injury and intrahepatic cholestasis. After the modelling was completed, the mice in each group were anesthetized, shaved, and disinfected. The abdominal cavity was opened, and the mice were quickly dissected. After removing the whole liver, they were rinsed with physiological saline. Fresh livers of some mice were stored in 4% paraformaldehyde overnight at 4°C for paraffin sectioning. The remaining livers were cut and put into liquid nitrogen and stored at −80°C in ultra‐low temperature refrigerator. All animal experiments approved by the Animal Care and Use Committee of Tongji University and were performed in accordance with the National Institutes of Health Guidelines.2.2A portion of the live tissue was fixed in 4% paraformaldehyde for at least 24 hour and then was subjected to dehydration and penetration. The specimen was then embedded in paraffin. The section was cut at a thickness of 4 μm for haematoxylin and eosin (H&E) staining and Masson's trichrome staining. Pathological changes were observed by light microscopy.2.3The prepared sections (4 μm) were heated at 60°C for 1 hour and then rehydrated with xylene and different concentrations of alcohol. The sections were washed three times with phosphate buffer solution (PBS) to block non‐specific binding sites, immersed in 5% bovine serum albumin (BSA) for 20 minutes at 37°C and then incubated for 10 minutes. Specimens were incubated overnight at 4°C with primary antibodies including Ck7 (1:200), Ck19 (1:200), AFP (1:500) and Lgr5 (1:100). On the second day, after incubation with a appropriate secondary antibody for 30 minutes at 37°C, the sections were analysed with a diaminobenzidine (DAB) kit to detect antibody binding. Finally, the slices were observed by optical microscopy.2.4The mice in the normal diet group and the DDC diet experimental group at the 4th week were selected, and the mice were sacrificed by intravenous injection of excess ether. We visualized the common bile duct (CHD), tied a suture around it and injected it with 1 mL of PBS with a small needle. Microfil™ was subsequently injected into the CHD until the IHBDs were filled with dye. The clearing protocol was performed per the Flow tech website with serial ethanol and methyl salicylate.2.5After the whole livers were dissected, finely diced, they were treated with dispose  in 2‐[4‐(hydroxyethyl)‐1‐piperazinyl] ethane sulphonic acid  and buffered DMEM (Gibco) for 60 minutes at 37°C.2.62.The isolated DRCs were cultured in DMEM culture medium (Gibco Industries) supplemented with 10% foetal bovine serum (Hyclone), 100 U/mL penicillin and 100 g/mL streptomycin (Gibco) in a humidified incubator at 37°C with 5% CO2) for 2 hours Except for Normal Control group, other group was treated with β‐catenin overexpression plasmid, empty plasmid and inhibitor shRNA, respectively. Cell viability was measured with the CCK‐8 assay at a wavelength of 450 nm by using a microplate reader for further half‐maximal inhibitory concentration (IC50) analysis.The cells were plated in 96‐well plates, and 10 µL CCK‐8 solution (Peptide Institute) was then added to each well. The plate was maintained in a dark incubator  for 1 hour at 4°C. The tissues were washed with PBS three times, then the tissues were dehydrated overnight in 30% sucrose (dissolved in PBS) at 4°C. The tissues were embedded in OCT (Sakura) for 2 hours on day 2 and stored at −80°C. 5 µm frozen sections were collected on slides. Before immunofluorescent staining, the tissue sections were dried at room temperature for 5 minutes and then put in PBS to dissolve OCT for 5 minutes. Next, tissue sections were ruptured with 5% PBST (containing 5% donkey serum in PBS and 0.1% Triton X‐100 in PBS) for 30 minutes at room temperature. The non‐specific antigen‐binding sites were blocked with 5% BSA, and the sections were then incubated with primary antibodies at 4°C overnight in dark. The slides incubated with secondary antibodies for 30 minutes at room temperature in dark on day 2 and then were incubated with 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride (DAPI) (Vector Laboratories). Olympus confocal (FV1200) was used to observed immunostaining images. Images were analysed by ImageJ (NIH) software.2.8β‐actin. The primer pairs used in our experiment are listed in Table The TRIzol reagent was used to extract total RNA. cDNA was synthesized using SuperScript II reverse transcriptase with Oligo . Real‐time PCR was carried out with a 7500 real‐time PCR system (Applied Biosystems) using the SYBR Green PCR Kit (Takara Japan) according to manufacturers’ instructions. The levels of the target genes were normalized to 2.9β‐actin (1:1000), Ck7 (1:200), Ck19 (1:200), AFP (1:500), Lgr5 (1:100), β‐actin was used as an internal control. On the second day, the membranes were washed with PBST three times and then incubated with the secondary antibody horseradish peroxidase‐conjugated anti‐rabbit or anti‐mouse IgG (1:2000) for 1 hour at 37°C. After three washes, the membranes were scanned using the Odyssey two‐colour infrared laser imaging system (Licor).Total cellular proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer (Kaiji Biology) containing protease inhibitors, and the protein concentration was detected with a BCA kit (Kaiji Biology). Equal amounts of protein samples (20 µg) were resolved by SDS‐polyacrylamide gel electrophoresis and transferred to polyvinyl difluoride (PVDF) membranes (Millipore Corp). The membranes were sequentially blocked with 5% BSA dissolved in phosphate buffered saline containing 0.1% Tween‐20 (PBST) to block non‐specific binding sites for 1 hour, followed by incubation in monoclonal primary antibodies overnight at 4°C. The primary antibodies were diluted as follows: 2.10t test. A one‐way ANOVA was used when more than two groups were being compared, a two‐way ANOVA was used when two groups were split between two independent variables, and a two‐tailed Mann‐Whitney U test was used when n was too small to determine normal distribution or the data were non‐parametric. F tests were used to compare variances between groups. Values of *P < .05, **P < .01 and ***P < .001 were considered statistically significant.All data were collected from at least three independent experiments as indicated. GraphPad Prism Software version 6.0 for Windows (GraphPad) was used for all statistical analysis. All data are presented as mean values ± SEM. For compare data, data significance was analysed using a two‐tailed unpaired Student's 33.1We observed the pathological changes of liver tissue through HE staining, it was found that compared with the normal diet group, the DDC model group showed obvious liver fibrosis, extracellular matrix deposition, cholestasis and proliferation of the small original tube‐like structure around the bile duct Figure . We re‐eThese new primitive tube‐like structures are very similar to the biological shape of the bile duct. We further detected by immunohistochemical staining and found that compared with the normal diet group, cholangiocytes surface markers CK7 and hepatic progenitor cells surface marker EpCAM in the DDC diet model group increased significantly, proving the existence of regeneration of neonatal bile duct cells, suggesting that these original tube‐like structures are new small bile ducts, which are composed of DRCs Figure .3.2To further study the biological characteristics of regenerated DRCs, we isolated it from DDC diet model (4 weeks) by two‐step collagenase lavage method, immunomagnetic bead method and density gradient centrifugation Figure . After c3.3P < .05)  Figure . After f) Figure .3.4β‐catenin overexpression on the proliferation ability of DRCs. The β‐catenin overexpression plasmid, empty plasmid and shRNA were transferred into DRCs respectively. By comparing the cell proliferation activity of the four groups, the experimental results suggested that after the β‐catenin overexpression plasmid was transferred into DRCs, compared with the empty plasmid group and the untreated negative control group, the proliferation activity of DRCs was significantly enhanced, while the transfer of shRNA significantly reduced the proliferation activity of DRCs. These above results indicate that in neonatal DRCs, the transfer of β‐catenin can promote the proliferation of DRCs cells, but after the transfer of shRNA, the proliferation of DRCs cells will be reduced, this difference is statistically significant (P < .05)  Figure .β‐catenin is involved in the process of bile duct injury and repair by regulating the proliferation and differentiation of DRCs, we transferred β‐catenin overexpression plasmid and shRNA in DRCs cultured in vitro to detect bile duct specific marker expression of CK7, CK19 and hepatic cell surface marker AFP. Through Western blot and real‐time fluorescent PCR experiments, it was found that at the protein and mRNA levels, the expression of CK7 and CK19 in DRCs was enhanced compared with the untreated control group and was significantly enhanced compared with the shRNA inhibition group. While the expression of AFP in DRCs was obviously weaker than that in untreated control group, and was significantly enhanced compared with shRNA suppression group  can be regenerated, bile duct cells can also promote liver regeneration when hepatocyte regeneration is impaired.DR is a common injury response in a variety of cholestasis and other liver diseases.In DR studies, EpCAM and SOX9 are often identified as stem cell markers.β‐catenin signalling.Studies have shown that many intercellular signalling pathways, such as Wnt, HGF, BMP and Notch, have been shown to be involved in the development of the liver, especially the bile duct,β‐catenin signal is related to bile duct injury repair by regulating the differentiation of DRCs. In vitro experiments we verified that β‐catenin has a positive regulatory effect on DRCs, suggesting that the Wnt signalling pathway can regulate the differentiation of neonatal DRCs, supporting the significance of Wnt/β‐catenin signalling in the repair of bile duct injury. Although the relevance of DRCs for promoting bile duct regeneration, the mechanism regulates this process is not fully understood. Further research is needed to elucidate the more detail of Wnt/β‐catenin signalling in DRCs. We encourage more work on these processes, which will hopefully lead to better therapeutic strategies for curing cholangiopathies.Since DRCs have the potential of stem cell bidirectional differentiation and share biological functions with embryonic liver progenitor cells, these results urge us to study whether Wnt/β‐catenin signalling pathway and its regulatory mechanisms involved in intrahepatic bile duct regeneration through DRCs proliferation, but also provides a new potential research target for hepatobiliary diseases. However, a more comprehensive understanding of this mechanism between DRCs and Wnt/β‐catenin signalling in biliary duct regeneration and repair is still unclear. We hope to make further efforts in future work, with a view to bringing more effective treatment for cholangiopathies.In conclusion, this study not only expands our understanding of the Wnt/The authors declare no conflict of interest, financial or otherwise.Nan Wang: Data curation (lead); Formal analysis (lead); Investigation (supporting); Methodology (supporting); Project administration (supporting). Rui Kong: Data curation (supporting); Formal analysis (supporting); Methodology (supporting). Wei Han: Investigation (supporting). Jie Lu: Conceptualization (lead); Funding acquisition ; Project administration (lead).