Establishment of a cholangiocarcinoma risk evaluation model based on mucin expression levels

BACKGROUND Cholangiocarcinoma(CCA)is a highly malignant cancer,characterized by frequent mucin overexpression.MUC1 has been identified as a critical oncogene in the progression of CCA.However,the comprehensive understanding of how the mucin family influences CCA progression and prognosis is still incomplete.AIM To investigate the functions of mucins on the progression of CCA and to establish a risk evaluation formula for stratifying CCA patients.METHODS Single-cell RNA sequencing data from 14 CCA samples were employed for elucidating the roles of mucins,complemented by bioinformatic analyses.Subse-quent validations were conducted through spatial transcriptomics and immuno-histochemistry.The construction of a risk evaluation model utilized the least absolute shrinkage and selection operator regression algorithm,which was further confirmed by independent cohorts and diverse data types.RESULTS CCA tumor cells with elevated levels of MUC1 and MUC4 showed activated nucleotide metabolic pathways and increased invasiveness.MUC5AC-high cells were found to promote CCA progression through WNT signaling.MUC5B-high cells exhibited robust cellular oxidation activities,leading to resistance against antitumoral treatments.MUC13-high cells were observed to secret chemokines,recruiting and transforming macrophages into the M2-polarized state,thereby suppressing antitumor immunity.MUC16-high cells were found to promote tumor progression through interleukin-1/nuclear factor kappa-light-chain-enhancer of activated B cells signaling upon interaction with neutrophils.Utilizing the expression levels of these mucins,a risk factor evaluation formula for CCA was developed and validated across multiple cohorts.CCA samples with higher risk factors exhibited stronger metastatic potential,chemotherapy resistance,and poorer prognosis.CONCLUSION Our study elucidates the functional mechanisms through which mucins contribute to CCA development,and provides tools for risk stratification in CCA.

Early identification of STEMI patients with emergency chest pain using lipidomics combined with machine learning

OBJECTIVES To analyze the differential expression of lipid spectrum between ST-segment elevated myocardial infarction(STEMI)and patients with emergency chest pain and excluded coronary artery disease(CAD),and establish the predictive model which could predict STEMI in the early stage.METHODS We conducted a single-center,nested case-control study using the emergency chest pain cohort of Peking Univer-sity Third Hospital.Untargeted lipidomics were conducted while LASSO regression as well as XGBoost combined with greedy algorithm were used to select lipid molecules.RESULTS Fifty-two STEMI patients along with 52 controls were enrolled.A total of 1925 lipid molecules were detected.There were 93 lipid molecules in the positive ion mode which were differentially expressed between the STEMI and the control group,while in the negative ion mode,there were 73 differentially expressed lipid molecules.In the positive ion mode,the differentially expressed lipid subclasses were mainly diacylglycerol(DG),lysophophatidylcholine(LPC),acylcarnitine(CAR),lysophospha-tidyl ethanolamine(LPE),and phosphatidylcholine(PC),while in the negative ion mode,significantly expressed lipid subclasses were mainly free fatty acid(FA),LPE,PC,phosphatidylethanolamine(PE),and phosphatidylinositol(PI).LASSO regression se-lected 22 lipids while XGBoost combined with greedy algorithm selected 10 lipids.PC(15:0/18:2),PI(19:4),and LPI(20:3)were the overlapping lipid molecules selected by the two feature screening methods.Logistic model established using the three lipids had excellent performance in discrimination and calibration both in the derivation set(AUC:0.972)and an internal validation set(AUC:0.967).In 19 STEMI patients with normal cardiac troponin,18 patients were correctly diagnosed using lipid model.CONCLUSIONS The differentially expressed lipids were mainly DG,CAR,LPC,LPE,PC,PI,PE,and FA.Using lipid molecules selected by XGBoost combined with greedy algorithm and LASSO regression to establish model could accurately predict STEMI even in the more earlier stage.

Angiogenesis in hepatocellular carcinoma:mechanisms and anti-angiogenic therapies

Hepatocellular carcinoma(HCC)is the fourth leading cause of cancer-associated death worldwide.Angiogenesis,the process of formation of new blood vessels,is required for cancer cells to obtain nutrients and oxygen.HCC is a typical hypervascular solid tumor with an aberrant vascular network and angiogenesis that contribute to its growth,progression,invasion,and metastasis.Current anti-angiogenic therapies target mainly tyrosine kinases,vascular endothelial growth factor receptor(VEGFR),and plateletderived growth factor receptor(PDGFR),and are considered effective strategies for HCC,particularly advanced HCC.However,because the survival benefits conferred by these anti-angiogenic therapies are modest,new anti-angiogenic targets must be identified.Several recent studies have determined the underlying molecular mechanisms,including pro-angiogenic factors secreted by HCC cells,the tumor microenvironment,and cancer stem cells.In this review,we summarize the roles of pro-angiogenic factors;the involvement of endothelial cells,hepatic stellate cells,tumor-associated macrophages,and tumor-associated neutrophils present in the tumor microenvironment;and the regulatory influence of cancer stem cells on angiogenesis in HCC.Furthermore,we discuss some of the clinically approved anti-angiogenic therapies and potential novel therapeutic targets for angiogenesis in HCC.A better understanding of the mechanisms underlying angiogenesis may lead to the development of more optimized anti-angiogenic treatment modalities for HCC.

The protocadherin alpha cluster is required for axon extension and myelination in the developing central nervous system

In adult mammals, axon regeneration after central nervous system injury is very poor, resulting in persistent functional loss. Enhancing the ability of axonal outgrowth may be a potential treatment strategy because mature neurons of the adult central nervous system may retain the intrinsic ability to regrow axons after injury. The protocadherin （Pcdh） clusters are thought to function in neuronal morphogenesis and in the assembly of neural circuitry in the brain. We cultured primary hippocampal neurons from E17.5 Pcdhα deletion （del-α） mouse embryos. After culture for 1 day, axon length was obviously shorter in del-α neurons compared with wild-type neurons. RNA sequencing of hippocampal E17.5 RNA showed that expression levels of BDNF, Fmod, Nrp2, OGN, and Sema3d, which are associated with axon extension, were significantly down-regulated in the absence of the Pcdhα gene cluster. Using transmission electron microscopy, the ratio of myelinated nerve fibers in the axons of del-α hippocampal neurons was significantly decreased; myelin sheaths of P21 Pcdhα-del mice showed lamellar disorder, discrete appearance, and vacuoles. These results indicate that the Pcdhα cluster can promote the growth and myelination of axons in the neurodevelopmental stage.

Evolving insights:how DNA repair pathways impact cancer evolution

Viewing cancer as a large,evolving population of heterogeneous cells is a common perspective.Because genomic instability is one of the fundamental features of cancer,this intrinsic tendency of genomic variation leads to striking intratumor heterogeneity and functions during the process of cancer formation,development,metastasis,and relapse.With the increased mutation rate and abundant diversity of the gene pool,this heterogeneity leads to cancer evolution,which is the major obstacle in the clinical treatment of cancer.Cells rely on the integrity of DNA repair machineries to maintain genomic stability,but these machineries often do not function properly in cancer cells.The deficiency of DNA repair could contribute to the generation of cancer genomic instability,and ultimately promote cancer evolution.With the rapid advance of new technologies,such as single-cell sequencing in recent years,we have the opportunity to better understand the specific processes and mechanisms of cancer evolution,and让s relationship with DNA repair.Here,we review recent findings on how DNA repair affects cancer evolution,and discuss how these mechanisms provide the basis for critical clinical challenges and therapeutic applications.

The mechanism and therapy of aortic aneurysms

Aortic aneurysm is a chronic aortic disease affected by many factors.Although it is generally asymptomatic,it poses a significant threat to human life due to a high risk of rupture.Because of its strong concealment,it is difficult to diagnose the disease in the early stage.At present,there are no effective drugs for the treatment of aneurysms.Surgical intervention and endovascular treatment are the only therapies.Although current studies have discovered that inflammatory responses as well as the production and activation of various proteases promote aortic aneurysm,the specific mechanisms remain unclear.Researchers are further exploring the pathogenesis of aneurysms to find new targets for diagnosis and treatment.To better understand aortic aneurysm,this review elaborates on the discovery history of aortic aneurysm,main classification and clinical manifestations,related molecular mechanisms,clinical cohort studies and animal models,with the ultimate goal of providing insights into the treatment of this devastating disease.The underlying problem with aneurysm disease is weakening of the aortic wall,leading to progressive dilation.If not treated in time,the aortic aneurysm eventually ruptures.An aortic aneurysm is a local enlargement of an artery caused by a weakening of the aortic wall.The disease is usually asymptomatic but leads to high mortality due to the risk of artery rupture.

Deep learning facilitated whole live cell fast super-resolution imaging

A fully convolutional encoder-decoder network(FCEDN),a deep learning model,was developed and applied to image scanning microscopy(ISM).Super-resolution imaging was achieved with a 78μm×78μm field of view and 12.5 Hz-40 Hz imaging frequency.Mono and dual-color continuous super-resolution images of microtubules and cargo in cells were obtained by ISM.The signal-to-noise ratio of the obtained images was improved from 3.94 to 22.81 and the positioning accuracy of cargoes was enhanced by FCEDN from 15.83±2.79 nm to 2.83±0.83 nm.As a general image enhancement method,FCEDN can be applied to various types of microscopy systems.Application with conventional spinning disk confocal microscopy was demonstrated and significantly improved images were obtained.

DNMT3A/3B overexpression might be correlated with poor patient survival, hypermethylation and low expression of ESR1/PGR in endometrioid carcinoma: an analysis of The Cancer Genome Atlas

Background:DNA methylation is involved in numerous biologic events and associates with transcriptional gene silencing, playing an important role in the pathogenesis of endometrial cancer.ESR1/PGR frequently undergoes de novo methylation and loss expression in a wide variety of tumors, including breast, colon, lung, and brain tumors.However, the mechanisms underlying estrogen and progesterone receptors (ER/PR) loss in endometrial cancer have not been studied extensively.The aims of this study were to determine the expression of DNA (cytosine-5)-methyltransferase 3A/3B (DNMT3A/3B) in endometrial cancer to investigate whether the methylation catalyzed by DNMT3A/3B contributes to low ER/PR expression.Methods:The clinicopathologic information and RNA-Seq expression data of DNMT3A/3B of 544 endometrial cancers were derived from The Cancer Genome Atlas (TCGA) uterine cancer cohort in May 2018.RNA-Seq level of DNMT3A/3B was compared between these clinicopathologic factors with t-test or one-way analysis of variance.Results:DNMT3A/3B was overexpressed in endometrioid carcinoma (EEC) and was even higher in non-endometrioid carcinoma (NEEC) (DNMT3A, EEC vs.NEEC:37.6% vs.69.9%, t=-7.440, P<0.001;DNMT3B, EEC vs.NEEC:42.4% vs.72.8 %, t=-6.897, P<0.001).In EEC, DNMT3A overexpression was significantly correlated with the hypermethylation and low expression of the ESR1 and PGR (P<0.05).The same trend was observed in the DNMT3B overexpression subgroup.In the ESR1/PGR low-expression subgroups, as much as 83.1% of ESR1 and 59.5% of PGR were hypermethylated, which was significantly greater than the ESR1/PGR high-expression subgroups (31.3% and 11.9%, respectively).However, the above phenomena were absent in NEEC, while DNMT3A/3B overexpression, ESR1/PGR hypermethylation, and low ER/PR expression occurred much more often.In univariate analysis, DNMT3A/3B overexpressions were significantly correlated with worse prognosis.In multivariate analysis, only DNMT3A was an independent predictor of disease-free survival (P<0.05).Conclusions:DNMT3A/3B expression increases progressively from EEC to NEEC and is correlated with poor survival.The mechanisms underlying low ER/PR expression might be distinct in EEC vs.NEEC.In EEC, methylation related to DNMT3A/3B overexpression might play a major role in ER/PR downregulation.

Deacetylation of TFEB promotes fibrillar Aβ degradation by upregulating lysosomal biogenesis in microglia

Microglia play a pivotal role in clearance of Aβ by degrading them in lysosomes, countering amyloid pla- que pathogenesis in Alzheimer＇s disease （AD）. Recent evidence suggests that lysosomal dysfunction leads to insufficient elimination of toxic protein aggregates. We tested whether enhancing lysosomal function with transcription factor EB （TFEB）, an essential regulator modulating lysosomal pathways, would promote Aβ clearance in microglia. Here we show that microglial expression of TFEB facilitates fibrillar Aβ （fAβ） degra- dation and reduces deposited amyloid plaques, which are further enhanced by deacetylation of TFEB. Using mass spectrometry analysis, we firstly confirmed acetylation as a previously unreported modification of TFEB and found that SIRT1 directly interacted with and deacetylated TFEB at lysine residue 116. Subsequently, SIRT1 overexpression enhanced lysosomal function and fAβ degradation by upregulating transcriptional levels of TFEB downstream targets, which could be inhibited when TFEB was knocked down. Furthermore, overexpression of deacetylated TFEB at K116R mutant in microglia accelerated intracellular fAβ degradation by stimulating lysosomal biogenesis and greatly reduced the deposited amyloid plaques in the brain slices of APPIPS1 transgenic mice. Our findings reveal that deacetylaUon of TFEB could regulate lysosomal biogenesis and fAβ degradation, making microglial activation of TFEB a possible strategy for attenuating amyloid plaque deposition in AD.

Role of farnesoid X receptor in establishment of ontogeny of phase-I drug metabolizing enzyme genes in mouse liver

The expression of phase-I drug metabolizing enzymes in liver changes dramatically during postnatal liver maturation.Farnesoid X receptor(FXR) is critical for bile acid and lipid homeostasis in liver.However,the role of FXR in regulating ontogeny of phase-I drug metabolizing genes is not clear.Hence,we applied RNA-sequencing to quantify the developmental expression of phase-I genes in both Fxr-null and control(C57BL/6) mouse livers during development.Liver samples of male C57BL/6 and Fxr-null mice at6 different ages from prenatal to adult were used.The Fxr-null showed an overall effect to diminish the "day-1 surge" of phase-I gene expression,including cytochrome P450 s at neonatal ages.Among the 185 phase-I genes from 12 different families,136 were expressed,and differential expression during development occurred in genes from all 12 phase-I families,including hydrolysis: carboxylesterase(Ces),paraoxonase(Pon),and epoxide hydrolase(Ephx); reduction: aldoketo reductase(Akr),quinone oxidoreductase(Nqo),and dihydropyrimidine dehydrogenase(Dpyd); and oxidation: alcohol dehydrogenase(Adh),aldehyde dehydrogenase(Aldh),flavin monooxygenases(Fmo),molybdenum hydroxylase(Aox and Xdh),cytochrome P450(P450),and cytochrome P450 oxidoreductase(Por).The data also suggested new phase-I genes potentially targeted by FXR.These results revealed an important role of FXR in regulation of ontogeny of phase-I genes.

Epigenetic regulation of drug metabolism and transport

The drug metabolism is a biochemical process on modification of pharmaceutical substances through specialized enzymatic systems. Changes in the expression of drug-metabolizing enzyme genes can affect drug metabolism. Recently, epigenetic regulation of drug-metabolizing enzyme genes has emerged as an important mechanism. Epigenetic regulation refers to heritable factors of genomic modifications that do not involve changes in DNA sequence. Examples of such modifications include DNA methylation, histone modifications, and non-coding RNAs. This review examines the widespread effect of epigenetic regulations on genes involved in drug metabolism, and also suggests a network perspective of epigenetic regulation. The epigenetic mechanisms have important clinical implications and may provide insights into effective drug development and improve safety of drug therapy.

Many facades of CTCF unified by its coding for three-dimensional genome architecture

CCCTC-binding factor(CTCF)is a multifunctional zinc finger protein that is conserved in metazoan species.CTCF is consistently found to play an important role in many diverse biological processes.CTCF/cohesin-mediated active chromatin‘loop extrusion’architects three-dimensional(3D)genome folding.The 3D architectural role of CTCF underlies its multifarious functions,including developmental regulation of gene expression,protocadherin(Pcdh)promoter choice in the nervous system,immunoglobulin(Ig)and T-cell receptor(Tcr)V(D)J recombination in the immune system,homeobox(Hox)gene control during limb development,as well as many other aspects of biology.Here,we review the pleiotropic functions of CTCF from the perspective of its essential role in 3D genome architecture and topological promoter/enhancer selection.We envision the 3D genome as an enormous complex architecture,with tens of thousands of CTCF sites as connecting nodes and CTCF proteins as mysterious bonds that glue together genomic building parts with distinct articulation joints.In particular,we focus on the internal mechanisms by which CTCF controls higher order chromatin structures that manifest its many fa?ades of physiological and pathological functions.We also discuss the dichotomic role of CTCF sites as intriguing3D genome nodes for seemingly contradictory‘looping bridges’and‘topological insulators’to frame a beautiful magnificent house for a cell’s nuclear home.

Genetic evidence for asymmetric blocking of higher-order chromatin structure by CTCF/cohesin

Dear Editor,Similar to higher-order folding of polypeptide chains into functional proteins,linear DNA molecules are spatially folded in a hierarchical and dynamic manner into three-dimensional(3D)functional chromatin structures in eukaryotic nuclei(Huang and Wu,2016;Rowley and Corces,2018).This dynamic folding is closely related to many nuclear processes such as DNA replication and repair,chromosomal translo­cation,recombination,and segregation,as well as RNA transcription,splicing,and transport.In particular,dynamic long-distance chromatin looping interactions,which result in close spatial contacts between distal enhancers and target promoters,are thought to play a role in controlling precise spatiotemporal as well as cell-type specific gene expression during animal development(Rowley and Corces,2018).Mammalian genomes contain numerous noncoding regula­tory elements that regulate these dynamic long-distance chromatin looping interactions.

Increased Expression of Colonic Mucosal Melatonin in Patients with Irritable Bowel Syndrome Correlated with Gut Dysbiosis

Dysregulation of the gut microbiota/gut hormone axis contributes to the pathogenesis of irritable bowel syndrome(IBS).Melatonin plays a beneficial role in gut motility and immunity.However,altered expression of local mucosal melatonin in IBS and its relationship with the gut microbiota remain unclear.Therefore,we aimed to detect the colonic melatonin levels and microbiota profiles in patients with diarrhea-predominant IBS(IBS-D)and explore their relationship in germ-free(GF)rats and BON-1 cells.Thirty-two IBS-D patients and twenty-eight healthy controls(HCs)were recruited.Fecal specimens from IBS-D patients and HCs were separately transplanted into GF rats by gavage.The levels of colon mucosal melatonin were assessed by immunohistochemical methods,and fecal microbiota communities were analyzed using 16S rDNA sequencing.The effect of butyrate on melatonin synthesis in BON-1 cells was evaluated by ELISA.Melatonin levels were significantly increased and negatively correlated with visceral hypersensitivity in IBS-D patients.GF rats inoculated with fecal microbiota from IBS-D patients had high colonic melatonin levels.Butyrate-producing Clostridium cluster XIVa species,such as Roseburia species and Lachnospira species,were positively related to colonic mucosal melatonin expression.Butyrate significantly increased melatonin secretion in BON-1 cells.Increased melatonin expression may be an adaptive protective mechanism in the development of IBS-D.Moreover,some Clostridium cluster XIVa species could increase melatonin expression via butyrate production.Modulation of the gut hormone/gut microbiota axis offers a promising target of interest for IBS in the future.

Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9

The human genome contains millions of DNA regulatory elements and a large number of gene clusters,most of which have not been tested experimentally.The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)programed with a synthetic single-guide RNA(sgRNA)emerges as a method for genome editing in virtually any organisms.Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs.Specifically,we found that,in cultured human cells and mice,efficient precise inversions of DNA fragments ranging in size froma few tens of bp to hundreds of kb could be generated.In addition,DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks(DSBs)on two homologous chromosomes(chromatids).Moreover,junctions of combinatorial inversions and duplications of the protocadherin(Pcdh)gene clusters induced by Cas9 with four sgRNAs could be detected.In mice,we obtained founders with alleles of precise inversions,duplications,and deletions of DNA fragments of variable sizes by CRISPR.Interestingly,we found that very efficient inversions were mediated by microhomology-mediated end joining(MMEJ)through short inverted repeats.We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice.Finally,we applied this CRISPR method to a regulatory element of the Pcdha cluster and found a new role in the regulation of members of the Pcdhg cluster.This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters.

Restoration of electrical microenvironment enhances bone regeneration under diabetic conditions by modulating macrophage polarization

Macrophage-mediated inflammation compromises bone repair in diabetic patients.Electrical signaling cues are known to regulate macrophage functions.However,the biological effects of electrical microenvironment from charged biomaterials on the immune response for regulating osteogenesis under diabetic conditions remain to be elucidated.Herein the endogeneous electrical microenvironment of native bone tissue was recapitulated by fabricating a ferroelectric BaTiO_(3)/poly(vinylidene fluoridetrifluoroethylene)(BTO/P(VDF-TrFE))nanocomposite membrane.In vitro,the polarized BaTiO_(3)/poly(vinylidene fluoridetrifluoroethylene)(BTO/P(VDF-TrFE))nanocomposite membranes inhibited high glucose-induced M1-type inflammation,by effecting changes in cell morphology,M1 marker expression and pro-inflammatory cytokine secretion in macrophages.This led to enhanced osteogenic differentiation of human bone marrow mesenchymal stem cells(BM-MSCs).In vivo,the biomimetic electrical microenvironment recapitulated by the polarized nanocomposite membranes switched macrophage phenotype from the pro-inflammatory(M1)into the pro-healing(M2)phenotype,which in turn enhanced bone regeneration in rats with type 2 diabetes mellitus.Mechanistic studies revealed that the biomimetic electrical microenvironment attenuated pro-inflammatory M1 macrophage polarization under hyperglycemic conditions by suppressing expression of AKT2 and IRF5 within the PI3K-AKT signaling pathway,thereby inducing favorable osteo-immunomodulatory effects.Our study thus provides fundamental insights into the biological effects of restoring the electrical microenvironment conducive for osteogenesis under DM conditions,and offers an effective strategy to design functionalized biomaterials for bone regeneration therapy in diabetic patients.

CRISPR Double Cutting through the Labyrinthine Architecture of 3D Genomes

The genomes are organized into ordered and hierarchical topological structures in interphase nuclei.Within discrete territories of each chromosome,topologically associated domains（TADs） play important roles in various nuclear processes such as gene regulation.Inside TADs separated by relatively constitutive boundaries,distal elements regulate their gene targets through specific chromatin-looping contacts such as long-distance enhancer-promoter interactions.High-throughput sequencing studies have revealed millions of potential regulatory DNA elements,which are much more abundant than the mere ~ 20,000 genes they control.The recently emerged CRISPRCas9 genome editing technologies have enabled efficient and precise genetic and epigenetic manipulations of genomes.The multiplexed and high-throughput CRISPR capabilities facilitate the discovery and dissection of gene regulatory elements.Here,we describe the applications of CRISPR for genome,epigenome,and 3D genome editing,focusing on CRISPR DNA-fragment editing with Cas9 and a pair of sgRNAs to investigate topological folding of chromatin TADs and developmental gene regulation.

Facile Generation of Strained Peptidyl Thiolactones from Hydrazides and Its Application in Assembling MUC-1 VNTR OIigomers

An efficient method for the activation of C-terminal 4-mecaptoproline-or penicillamine-containing peptide hydrazides in ligation re-actions is reported herein.The corresp on ding peptide hydrazides can be readily prepared using solid-phase peptide synthesis,and subsequently activated by acetylacet one(acac)without exoge nous thiol additives.Strained peptidyl thiolactones could be the possible reactive in termediates that drastically accelerate the reacti on rates at the sterically demandi ng proline and valine sites.This developed protocol allows for sequential peptide ligations in a one-pot manner,and expedites the assembly of mucin 1(MUC-1)variable number tandem repeat(VNTR)trimers in various glycosylated forms.

Loss of 4.1N in epithelial ovarian cancer results in EMT and matrix-detached cell death resistance

Epithelial ovarian cancer(EOC)is one of the leading causes of death from gynecologic cancers and peritoneal dissemination is the major cause of death in patients with EOC.Although the loss of 4.1N is associated with increased risk of malignancy,its association with EOC remains unclear.To explore the underlying mechanism of the loss of 4.1N in constitutive activation of epithelial-mesenchymal transition(EMT)and matrixdetached cell death resistance,we investigated samples from 268 formalin-fixed EOC tissues and performed various in vitro and in vivo assays.We report that the loss of 4.1N correlated with progress in clinical stage,as well as poor survival in EOC patients.The loss of 4.1N induces EMT in adherent EOC cells and its expression inhibits anoikis resistance and EMT by directly binding and accelerating the degradation of 14-3-3 in suspension EOC cells.Furthermore,the loss of 4.1N could increase the rate of entosis,which aggravates cell death resistance in suspension EOC cells.Moreover,xenograft tumors in nude mice also show that the loss of 4.1N can aggravate peritoneal dissemination of EOC cells.Single-agent and combination therapy with a ROCK inhibitor and a 14-3-3 antagonist can reduce tumor spread to varying degrees.Our results not only define the vital role of 4.1N loss in inducing EMT,anoikis resistance,and entosis-induced cell death resistance in EOC,but also suggest that individual or combined application of 4.1N,14-3-3 antagonists,and entosis inhibitors may be a promising therapeutic approach for the treatment of EOC.

Proteomic analysis and miRNA profiling of human testicular endothelial cell-derived exosomes:the potential effects on spermatogenesis

Testicular endothelial cells have been found to play an important role in spermatogenesis and fertility,but their mechanism is obscure.Exosomes released by various cells are recognized as cell–cell communication mediators during the initiation and progression of many diseases.Therefore,the current study aimed to investigate the protein and miRNA components of human testicular endothelial cell-derived exosomes(HTEC-Exos)and to explore their potential effects on spermatogenesis.In this study,HTEC-Exos were first isolated by the ultracentrifugation method,and then identified by nanoparticle tracking analysis,transmission electron microscopy(TEM),and western blotting.The characteristics of HTEC-Exos were examined by liquid chromatography–mass spectrometry and microRNA(miRNA)chip analysis.Bioinformatics analysis was performed to explore the potential role of the exosomal content on spermatogenesis.A total of 945 proteins were identified,11 of which were closely related to spermatogenesis.A total of 2578 miRNAs were identified.Among them,30 miRNAs demonstrated potential associations with male reproductive disorders,such as azoospermia,and spermatogenesis disorders.In particular,11 out of these 30 miRNAs have been proven to be involved in spermatogenesis based on available evidence.This study provides a global view of the proteins and miRNAs from HTEC-Exos,suggesting that HTEC-Exos may function as potential effectors during the process of spermatogenesis.