Transcriptome sequencing reveals novel biomarkers and immune cell infiltration in esophageal tumorigenesis

BACKGROUND Esophageal squamous cell carcinoma(ESCC)is one of the most common malignancies worldwide,and its development comprises a multistep process from intraepithelial neoplasia(IN)to carcinoma(CA).However,the critical regulators and underlying molecular mechanisms remain largely unknown.AIM To explore the genes and infiltrating immune cells in the microenvironment that are associated with the multistage progression of ESCC to facilitate diagnosis and early intervention.METHODS A mouse model mimicking the multistage development of ESCC was established by providing warter containing 4-nitroquinoline 1-oxide(4NQO)to C57BL/6 mice.Moreover,we established a control group without 4NQO treatment of mice.Then,transcriptome sequencing was performed for esophageal tissues from patients with different pathological statuses,including low-grade IN(LGIN),high-grade IN(HGIN),and CA,and controlled normal tissue(NOR)samples.Differentially expressed genes(DEGs)were identified in the LGIN,HGIN,and CA groups,and the biological functions of the DEGs were analyzed via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses.The CIBERSORT algorithm was used to detect the pattern of immune cell infilt-ration.Immunohistochemistry(IHC)was also conducted to validate our results.Finally,the Luminex multiplex cytokine analysis was utilized to measure the serum cytokine levels in the mice.RESULTS Compared with those in the NOR group,a total of 681541,and 840 DEGs were obtained in the LGIN,HGIN,and CA groups,respectively.Using the intersection of the three sets of DEGs,we identified 86 genes as key genes involved in the development of ESCC.Enrichment analysis revealed that these genes were enriched mainly in the keratinization,epidermal cell differentiation,and interleukin(IL)-17 signaling pathways.CIBERSORT analysis revealed that,compared with those in the NOR group,M0 and M1 macrophages in the 4NQO group showed stronger infiltration,which was validated by IHC.Serum cytokine analysis revealed that,compared with those in the NOR group,IL-1βand IL-6 were upregulated,while IL-10 was downregulated in the LGIN,HGIN,and CA groups.Moreover,the expression of the representative key genes,such as S100a8 and Krt6b,was verified in external human samples,and the results of immunohistochemical staining were consistent with the findings in mice.CONCLUSION We identified a set of key genes represented by S100a8 and Krt6b and investigated their potential biological functions.In addition,we found that macrophage infiltration and abnormal alterations in the levels of inflam-mation-associated cytokines,such as IL-1β,IL-6,and IL-10,in the peripheral blood may be closely associated with the development of ESCC.

Hepatic failure caused by plasma cell infiltration in multiple myeloma

Although plasma cell infiltration is not rare in autopsy of patients with multiple myeloma （MM）, it is very rarely detected in living patients. This is because MM rarely causes significant liver dysfunction that requires further evaluation. A 49-year-old man presented with acute renal failure and was diagnosed with kappa light chain MM stageⅡB. Thalidomide and dexamethasone were initiated. The patient developed a continuous increase in bilirubin that led to severe cholestasis. A liver biopsy revealed plasma cell infiltration. He then rapidly progressed to liver failure and died. Treatment options are limited in MM with significant liver dysfunction. Despite new drug therapies in MM, those patients with rapidly progressive liver failure appear to have a dismal outcome.

Hub genes associated with immune cell infiltration in breast cancer, identified through bioinformatic analyses of multiple datasets

Objective:The aim of this study was to identify hub genes associated with immune cell infiltration in breast cancer through bioinformatic analyses of multiple datasets.Methods:Nonparametric(NOISeq)and robust rank aggregation-ranked parametric(EdgeR)methods were used to assess robust differentially expressed genes across multiple datasets.Protein-protein interaction network,GO,KEGG enrichment,and subnetwork analyses were performed to identify immune-associated hub genes in breast cancer.Immune cell infiltration was evaluated with the CIBERSORT,XCELL,and TIMER methods.The association between the hub gene-based risk signature and survival was determined through Kaplan–Meier survival analysis,multivariate Cox analysis,and a nomogram with external verification.Results:We identified 163 robust differentially expressed genes in breast cancer through applying both nonparametric and parametric methods to multiple GEO(n=2,212)and TCGA(n=1,045)datasets.Integrated bioinformatic analyses further identified 10 hub genes:CXCL10,CXCL9,CXCL11,SPP1,POSTN,MMP9,DPT,COL1A1,ADAMDEC1,and RGS1.The 10 hub-gene-based risk signature significantly correlated with the prognosis of patients with breast cancer.Moreover,these hub genes were strongly associated with the extent of infiltration of CD4+T cells,CD8+T cells,neutrophils,macrophages,and myeloid dendritic cells into breast tumors.Conclusions:Integrated analyses of multiple databases led to the discovery of 10 robust hub genes that together may serve as a risk factor characteristic of the immune microenvironment in breast cancer.

Prognostic role of ring finger and WD repeat domain 3 and immune cell infiltration in hepatocellular carcinoma

We have found that the expression of ring finger and WD repeat domain 3(RFWD3)is significantly higher in unpaired and paired hepatocellular carcinoma(HCC)tissues than in normal tissues.Moreover,this expression has a significant correlation with the infiltration level of 14 immune cell types and when the detected RFWD3 expression levels were grouped as high and low,a prominent difference was revealed for overall survival,disease-specific survival,and progression-free interval.Through statistical analysis(univariate Cox),we were also able to identify RFWD3 as an independent prognostic element for HCC,with RFWD3 having an ability to accurately predict HCC prognosis(area under the curve of 0.863).Finally,we have generated prognostic nomograms for probabilities of 1-,3-and 5-year overall survival in HCC via integrating the factors of age,pathologic stage,alpha-fetoprotein level,and RFWD3 expression.

Study on the relationship between immune cell infiltration pattern and clinical characteristics and prognosis of cervical cancer based on TCGA database

Objective:To investigate the correlation between immune cell infiltration pattern and clinical features and prognosis of cervical carcinoma.Methods:All cervical cancer transcript data and related clinical data were downloaded from the public database Cancer Genome Atlas(TCGA),and the relative proportions of 22 invasive immune cell types were calculated by Cibersort software.Perl was used to assess the correlation between the pattern of immune cell invasion and clinical characteristics(age,clinical stage,tumor grade)in cervical cancer,and the correlation between the pattern of immune cell invasion and survival in cervical cancer was calculated by the K-M Log-Rank method.Result:The distribution of immune cells in 306 cases of cervical cancer and 3 cases of normal tissues was assessed using Cibersort.Compared with normal tissues,the contents of resting dendritic cells,activated dendritic cells,M1 macrophages and activated CD4+memory T cells were higher;the contents of M2 macrophages,neutrophils,regulatory T cells and activated mast cells were lower in cervical cancer tissues.The contents of M1 macrophages,unactivated CD4+memory T cells,andγδT cells were positively correlated with patient age(P<0.05).The contents of follicular helper T cells,activated and unactivated natural killer(NK)cells,and naive CD4 T cells were negatively correlated with patient age(P<0.05).Those with high resting dendritic cell composition had shorter overall survival,while those with high follicular helper T cell composition had longer overall survival(P<0.05).Conclusion:Compared with normal tissues,the composition of immune cells in cervical cancer tissues has certain specificity,which can provide reference for the early screening and diagnosis of the disease.Patients in different age groups may have different immune cell infiltration patterns,which can be used as a basis to explore drug targets in clinical practice.Resting dendritic cells and follicular helper T cells in cervical cancer can be used as possible efficacy predictors of clinical immunotherapy for cervical cancer.

Advancing neural regeneration via adaptable hydrogels:Enriched with Mg^(2+)and silk fibroin to facilitate endogenous cell infiltration and macrophage polarization

Peripheral nerve injury is a complex and challenging medical condition due to the limited ability of nerves to regenerate, resulting in the loss of both sensory and motor function. Hydrogels have emerged as a promising biomaterial for promoting peripheral nerve regeneration, while conventional hydrogels are generally unable to support endogenous cell infiltration due to limited network dynamics, thereby compromising the therapeutic outcomes. Herein, we present a cell adaptable hydrogel containing a tissue-mimetic silk fibroin network and a dynamically crosslinked bisphosphonated-alginate network. The dynamic network of this hydrogel can respond to cell-generated forces to undergo the cell-mediated reorganization, thereby effectively facilitating the rapid infiltration of Schwann cells and macrophages, as well as the ingrowth of axons. We further show that the magnesium ions released from the hydrogel not only promote neurite outgrowth but also regulate the polarization of macrophages in a sequential manner, contributing to the formation of a regenerative microenvironment. Therefore, this hydrogel effectively prevents muscle atrophy and promotes the regeneration and functional recovery of nerve defects of up to 10 mm within 8 weeks. The findings from this study demonstrate that adaptable hydrogels are promising inductive biomaterials for enhancing the therapeutic outcomes of peripheral nerve injury treatments.

Analysis and validation of diagnostic biomarkers and immune cell infiltration characteristics in pediatric sepsis by integrating bioinformatics and machine learning

Background Pediatric sepsis is a complicated condition characterized by life-threatening organ failure resulting from a dysregulated host response to infection in children.It is associated with high rates of morbidity and mortality,and rapid detection and administration of antimicrobials have been emphasized.The objective of this study was to evaluate the diagnostic biomarkers of pediatric sepsis and the function of immune cell infiltration in the development of this illness.Methods Three gene expression datasets were available from the Gene Expression Omnibus collection.First,the differentially expressed genes(DEGs)were found with the use of the R program,and then gene set enrichment analysis was carried out.Subsequently,the DEGs were combined with the major module genes chosen using the weighted gene co-expression network.The hub genes were identified by the use of three machine-learning algorithms:random forest,support vector machine-recursive feature elimination,and least absolute shrinkage and selection operator.The receiver operating characteristic curve and nomogram model were used to verify the discrimination and efficacy of the hub genes.In addition,the inflammatory and immune status of pediatric sepsis was assessed using cell-type identification by estimating relative subsets of RNA transcripts(CIBERSORT).The relationship between the diagnostic markers and infiltrating immune cells was further studied.Results Overall,after overlapping key module genes and DEGs,we detected 402 overlapping genes.As pediatric sepsis diagnostic indicators,CYSTM1(AUC=0.988),MMP8(AUC=0.973),and CD177(AUC=0.986)were investigated and demonstrated statistically significant differences(P<0.05)and diagnostic efficacy in the validation set.As indicated by the immune cell infiltration analysis,multiple immune cells may be involved in the development of pediatric sepsis.Additionally,all diagnostic characteristics may correlate with immune cells to varying degrees.Conclusions The candidate hub genes(CD177,CYSTM1,and MMP8)were identified,and the nomogram was constructed for pediatric sepsis diagnosis.Our study could provide potential peripheral blood diagnostic candidate genes for pediatric sepsis patients.

Enhancing cell infiltration of electrospun fibrous scaffolds in tissue regeneration

Electrospinning is one of the most effective approaches to fabricate tissue-engineered scaffolds composed of nano-to sub-microscale fibers that simulate a native extracellular matrix.However,one major concern about electrospun scaffolds for tissue repair and regeneration is that their small pores defined by densely compacted fibers markedly hinder cell infiltration and tissue ingrowth.To address this problem,researchers have developed and investigated various methods of manipulating scaffold structures to increase pore size or loosen the scaffold.These methods involve the use of physical treatments,such as salt leaching,gas foaming and custom-made collectors,and combined techniques to obtain electrospun scaffolds with loose fibrous structures and large pores.This article provides a summary of these motivating electrospinning techniques to enhance cell infiltration of electrospun scaffolds,which may inspire new electrospinning techniques and their new biomedical applications.

FTY720,Sphingosine 1-Phosphate Receptor Modulator,Ameliorates Experimental Autoimmune Encephalomyelitis by Inhibition of T Cell Infiltration

FTY720, a sphingosine 1-phosphate receptor modulator, induces a marked decrease in the number of peripheral blood lymphocytes and exerts immunomodulating activity in various experimental allograft and autoimmune disease models. In this study, we evaluated the effect of FTY720 and its active metabolite, （S）-enantiomer of FTY720-phosphate [（S）-FTY720-P] on experimental autoimmune encephalomyelitis （EAE） in rats and mice. Prophylactic administration of FTY720 at 0.1 to 1 mg/kg almost completely prevented the development of EAE, and therapeutic treatment with FTY720 significantly inhibited the progression of EAE and EAE-associated histological change in the spinal cords of LEW rats induced by immunization with myelin basic protein. Consistent with rat EAE, the development of proteolipid protein-induced EAE in SJL/J mice was almost completely prevented and infiltration of CD4^＋ T cells into spinal cord was decreased by prophylactic treatment with FTY720 and （S）-FTY720-P. When FTY720 or （S）-FTY720-P was given after establishment of EAE in SJL/J mice, the relapse of EAE was markedly inhibited as compared with interferon-β, and the area of demyelination and the infiltration of CD4^＋ T cells were decreased in spinal cords of EAE mice. Similar therapeutic effect by FTY720 was obtained in myelin oligodendrocyte glycoprotein-induced EAE in C57BL/6 mice. These results indicate that FTY720 exhibits not only a prophylactic but also a therapeutic effect on EAE in rats and mice, and that the effect of FTY720 on EAE appears to be due to a reduction of the infiltration of myelin antigen-specific CD4^＋ T cells into the inflammation site. Cellular ＆ Molecular Immunology. 2005;2（6）：439-448.

Identification of immune cell infiltration landscape for predicting prognosis of colorectal cancer

Background:The tumor microenvironment plays an essential role in the therapeutic response to immunotherapy.It is necessary to identify immune cell infiltration(ICI)subtypes for evaluating prognosis and therapeutic benefits.This study aimed to evaluate the ICI score as an effective prognostic biomarker for immune response.Methods:The cell-type identification by estimating relative subsets of RNA transcripts and the estimation of stromal and immune cells in malignant tumors using expression methods were used to analyse ICI landscapes in 161 colorectal cancer(CRC)samples with patients’clinical and prognostic data,RNA sequencing data,and whole-exome sequencing data from the Sixth Affiliated Hospital,Sun Yat-sen University(Guangzhou,China).Statistical analysis and data processing were conducted to calculate ICI scores,and to analyse the prognosis of CRC patients with different ICI scores and other features.A similar analysis with RNA sequencing and clinical data of colon adenocarcinoma(COAD)samples from The Cancer Genome Atlas(TCGA)database was conducted to confirm the correctness of the findings.Results:The high-ICI score group with a better prognosis(hazard ratio[HR],2.19;95%confidence interval[CI],1.03–4.64;logrank test,P=0.036)was characterized by the increased tumor mutational burden and interleukin-17(IL-17)signaling pathway.Significant differences in the prognosis and the expression levels of immune checkpoints and chemokine marker genes were found between the two ICI score groups.For COAD samples from TCGA,the results also showed a significant difference in patients’prognosis between the two ICI score groups(HR,1.72;95%CI,1.00–2.96;log-rank test,P=0.047).Conclusions:Tumor heterogeneity induced differences in identifying ICI subtypes of CRC patients.The ICI score may serve as an effective biomarker for predicting prognosis,help identify new therapeutic markers for CRC,and develop novel effective immune checkpoint blockade therapies.

Comprehensive analysis reveals hub genes associated with immune cell infiltration in allergic rhinitis

Objectives:Allergic rhinitis(AR)refers to a form of respiratory inflammation that mainly affects the sinonasal mucosa.The purpose of this study was to explore the level of immune cell infiltration and the pathogenesis of AR.Methods:We performed a comprehensive analysis of two gene expression profiles(GSE50223 and GSE50101,a total of 30 patients with AR and 31 healthy controls).CIBERSORT was used to evaluate the immune cell infiltration levels.Weighted gene coexpression network analysis was applied to explore potential genes or gene modules related to immune status,and enrichment analyses including gene ontology,Kyoto Encyclopedia of Genes and Genomes,gene set enrichment analysis,and gene set variation analysis,were performed to analyze the potential mechanisms in AR.A protein–protein interaction network was constructed to investigate the hub genes,and consensus clustering was conducted to identify the molecular subtypes of AR.Results:Compared to the healthy controls,patients with AR had high abundance levels and proportions of CD4+memory‐activated T cells.One hundred and eight immune‐related differentially expressed genes were identified.Enrichment analysis suggested that AR was mainly related to leukocyte cell‐cell adhesion,cytokine‐cytokine receptor interaction,T‐cell activation,and T‐cell receptor signaling pathway.Ten hub genes,includingTYROBP,CSF1R,TLR8,FCER1G,SPI1,ITGAM,CYBB,FCGR2A,CCR1,andHCK,which were related to immune response,might be crucial to the pathogenesis of AR.Three molecular subtypes with significantly different immune statuses were identified.Conclusion:This study improves our understanding of the molecular mechanisms in AR via comprehensive strategies and provides potential diagnostic biomarkers and therapeutic targets of AR.

Prognostic biomarkers and immune cell infiltration characteristics in small cell lung cancer

Background Small cell lung cancer(SCLC)is a highly malignant and aggressive neuroendocrine tumor.With the rise of immunotherapy,it has provided a new direction for SCLC.However,due to the lack of prognostic biomarkers,the median overall survival of SCLC is still to be improved.This study aimed to explore novel biomarkers and tumor-infiltrating immune cell characteristics that may serve as potential diagnostic and prognostic markers in SCLC.Methods Gene expression profiles from patients with SCLC were downloaded from the Gene Expression Omnibus(GEO)database,and tumor microenvironment(TME)infiltration profile data were obtained using CIBERSORT.The robust rank aggregation(RRA)method was utilized to integrate three SCLC microarray datasets downloaded from the GEO database and identify robust differentially expressed genes(DEGs)between normal and tumor tissue samples.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were performed to explore the functions of the robust DEGs.Subsequently,protein-protein interaction networks and key modules were constructed by Cytoscape,and hub genes were selected from the whole network using the plugin cytoHubba.Survival analysis of hub genes was performed by Kaplan-Meier plotter in 18 patients with extensive-stage SCLC.Results A total of 312 robust DEGs,including 55 upregulated and 257 downregulated genes,were screened from 129 SCLC tissue samples and 44 normal tissue samples.GO and KEGG enrichment analyses revealed that the robust DEGs were predominantly involved in human T-cell leukemia virus 1 infection,focal adhesion,complement and coagulation cascades,tumor necrosis factor(TNF)signaling pathway,and ECM-receptor interaction,which are closely associated with the development and progression of SCLC.Subsequently,three DEGs modules and six hub genes(ITGA10,DUSP12,PTGS2,FOS,TGFBR2,and ICAM1)were identified through screening with the Cytoscape plugins MCODE and cytoHubba,respectively.Immune cell infiltration analysis by the CIBERSORT algorithm revealed that resting memory CD4+T cells were the predominant infiltrating immune cells in SCLC.In addition,Kaplan-Meier plotter revealed that the gene prostaglandin-endoperoxide synthase 2(PTGS2)was a potential prognostic biomarker of SCLC.Conclusions Hub genes and tumor-infiltrating immune cells may be the molecular mechanisms underlying the development of SCLC,and this finding could contribute to the formulation of individualized immunotherapy strategies for SCLC.

B cell infiltration is highly associated with prognosis and an immune-infiltrated tumor microenvironment in neuroblastoma

Aim:Neuroblastoma is the most common extracranial solid tumor in children.Recent advances in immunotherapy Approaches,including in neuroblastoma,have shown the important role of the immune system in mounting an effective anti-tumor response.In this study,we aimed to provide a comprehensive investigation of immune cell infiltration in neuroblastoma utilizing a large number of gene expression datasets.Methods:We inferred immune cell infiltration using an established immune inference method and evaluated the association between immune cell abundance and patient prognosis as well as common chromosomal abnormalities found in neuroblastoma.In addition,we evaluated co-infiltration patterns among distinct immune cell types.Results:The infiltration of naïve B cells,NK cells,and CD8+T cells was associated with improved patient prognosis.Naïve B cells were the most consistent indicator of prognosis and associated with an active immune tumor microenvironment.Patients with high B cell infiltration showed high co-infiltration of other immune cell types and the enrichment of immune-related pathways.The presence of high B cell infiltration was associated with both recurrence-free and overall survival,even after adjusting for clinical variables.Conclusion:In this study,we have provided a comprehensive evaluation of immune cell infiltration in neuroblastoma using gene expression data.We propose an important role for B cells in the neuroblastoma tumor microenvironment and suggest that B cells can be used as a prognostic biomarker to predict recurrence-free and overall survival independently of currently utilized prognostic variables.

Identification of immune feature genes and intercellular profiles in diabetic cardiomyopathy

BACKGROUND Diabetic cardiomyopathy(DCM)is a multifaceted cardiovascular disorder in which immune dysregulation plays a pivotal role.The immunological molecular mechanisms underlying DCM are poorly understood.AIM To examine the immunological molecular mechanisms of DCM and construct diagnostic and prognostic models of DCM based on immune feature genes(IFGs).METHODS Weighted gene co-expression network analysis along with machine learning methods were employed to pinpoint IFGs within bulk RNA sequencing(RNA-seq)datasets.Single-sample gene set enrichment analysis(ssGSEA)facilitated the analysis of immune cell infiltration.Diagnostic and prognostic models for these IFGs were developed and assessed in a validation cohort.Gene expression in the DCM cell model was confirmed through real time-quantitative polymerase chain reaction and western blotting techniques.Additionally,single-cell RNA-seq data provided deeper insights into cellular profiles and interactions.RESULTS The overlap between 69 differentially expressed genes in the DCM-associated module and 2483 immune genes yielded 7 differentially expressed immune-related genes.Four IFGs showed good diagnostic and prognostic values in the validation cohort:Proenkephalin(Penk)and retinol binding protein 7(Rbp7),which were highly expressed,and glucagon receptor and inhibin subunit alpha,which were expressed at low levels in DCM patients(all area under the curves>0.9).SsGSEA revealed that IFG-related immune cell infiltration primarily involved type 2 T helper cells.High expression of Penk(P<0.0001)and Rbp7(P=0.001)was detected in cardiomyocytes and interstitial cells and further confirmed in a DCM cell model in vitro.Intercellular events and communication analysis revealed abnormal cellular phenotype transformation and signaling communication in DCM,especially between mesenchymal cells and macrophages.CONCLUSION The present study identified Penk and Rbp7 as potential DCM biomarkers,and aberrant mesenchymal-immune cell phenotype communication may be an important aspect of DCM pathogenesis.

A Prognostic Biomarker for Bladder Cancer Correlated with Immune Infiltration Is PAEP

Background: A major cause of cancer death worldwide is bladder cancer, which is the most common malignant tumor of the urinary tract. PAEP is a member of the kernel lipocalin superfamily whose members share relatively low sequence similarity but have highly conserved exon/intron structure and three-dimensional protein folding. Most lipocalins are clustered on the long arm of chromosome 9. The purpose of this study was to clarify the correlation between PAEP expression level and bladder cancer. Methods: In the TCGA database, we obtained clinical and RNA sequencing data of 431 BLCA patients, including 412 BLCA tissues and 19 normal bladder tissues in the study. Analyses of bioinformatics were conducted in this study to determine the role of PAEP in bladder cancer. A quantitative real-time PCR method was used to quantitate the gene expression profile. Additionally, the effect of PAEP on tumor immune infiltration and prognosis was analyzed. Results: PAEP was a poor prognostic biomarker of bladder cancer because it was significantly upregulated. bladder cancer patients with higher PAEP expression had poor outcomes. An AUC of 0.780 was calculated from the area under the ROC curve. PAEP was associated with T stage, pathologic stage, Histologic grade and Subtype of bladder cancer patients, and served as an independent predictor of overall survival in bladder cancer patients. Functional enrichment analysis revealed PAEP was obviously enriched in pathways connected with carcinogenesis and immunosuppression. The expression of PAEP was significantly associated with tumor immune cells and immune checkpoints according to ssGSEA and Spearman correlation analysis. Conclusions: In this study, we screened and detected a mRNA, PAEP is a prognostic and immune-related biomarker in BLCA, which may contribute to the early diagnosis and treatment of BLCA.

High spindle and kinetochore-associated complex subunit-3 expression predicts poor prognosis and correlates with adverse immune infiltration in hepatocellular carcinoma

BACKGROUND Spindle and kinetochore-associated complex subunit 3(SKA3)is a malignancyassociated gene that plays a critical role in the regulation of chromosome separation and cell division.However,the molecular mechanism through which SKA3 regulates tumor cell proliferation in hepatocellular carcinoma(HCC)has not been fully elucidated.AIM To investigate the molecular mechanisms underlying the role of SKA3 in HCC.METHODS SKA3 expression,clinicopathological,and survival analyses were performed using multiple public database platforms,and the results were verified by Western blot and immunohistochemistry staining using collected clinical samples.Functional enrichment analyses were performed to evaluate the biological functions and molecular mechanisms of SKA3 in HCC.Furthermore,the Tumor Immune Estimation Resource and single-sample Gene Set Enrichment Analysis(ssGSEA)algorithms were utilized to investigate the abundance of tumor-infiltrating immune cells in HCC.The response to chemotherapeutic drugs was evaluated by the R package“pRRophetic”.RESULTS We found that upregulated SKA3 expression was significantly correlated with poor prognosis in patients with HCC.Multivariable Cox regression analysis indicated that SKA3 was an independent risk factor for survival.GSEA revealed that SKA3 expression may facilitate proliferation and migratory processes by regulating the cell cycle and DNA repair.Moreover,patients with high SKA3 expression had significantly decreased ratios of CD8+T cells,natural killer cells,and dendritic cells.Drug sensitivity analysis showed that the high SKA3 group was more sensitive to sorafenib,sunitinib,paclitaxel,doxorubicin,gemcitabine,and vx-680.CONCLUSION High SKA3 expression led to poor prognosis in patients with HCC by enhancing HCC proliferation and repressing immune cell infiltration surrounding HCC.SKA3 may be used as a biomarker for poor prognosis and as a therapeutic target in HCC.

Action of circulating and infiltrating B cells in the immune microenvironment of colorectal cancer by single-cell sequencing analysis

BACKGROUND The complexity of the immune microenvironment has an impact on the treatment of colorectal cancer(CRC),one of the most prevalent malignancies worldwide.In this study,multi-omics and single-cell sequencing techniques were used to investigate the mechanism of action of circulating and infiltrating B cells in CRC.By revealing the heterogeneity and functional differences of B cells in cancer immunity,we aim to deepen our understanding of immune regulation and provide a scientific basis for the development of more effective cancer treatment strategies.AIM To explore the role of circulating and infiltrating B cell subsets in the immune microenvironment of CRC,explore the potential driving mechanism of B cell development,analyze the interaction between B cells and other immune cells in the immune microenvironment and the functions of communication molecules,and search for possible regulatory pathways to promote the anti-tumor effects of B cells.METHODS A total of 69 paracancer(normal),tumor and peripheral blood samples were collected from 23 patients with CRC from The Cancer Genome Atlas database(https://portal.gdc.cancer.gov/).After the immune cells were sorted by multicolor flow cytometry,the single cell transcriptome and B cell receptor group library were sequenced using the 10X Genomics platform,and the data were analyzed using bioinformatics tools such as Seurat.The differences in the number and function of B cell infiltration between tumor and normal tissue,the interaction between B cell subsets and T cells and myeloid cell subsets,and the transcription factor regulatory network of B cell subsets were explored and analyzed.RESULTS Compared with normal tissue,the infiltrating number of CD20+B cell subsets in tumor tissue increased significantly.Among them,germinal center B cells(GCB)played the most prominent role,with positive clone expansion and heavy chain mutation level increasing,and the trend of differentiation into memory B cells increased.However,the number of plasma cells in the tumor microenvironment decreased significantly,and the plasma cells secreting IgA antibodies decreased most obviously.In addition,compared with the immune microenvironment of normal tissues,GCB cells in tumor tissues became more closely connected with other immune cells such as T cells,and communication molecules that positively regulate immune function were significantly enriched.CONCLUSION The role of GCB in CRC tumor microenvironment is greatly enhanced,and its affinity to tumor antigen is enhanced by its significantly increased heavy chain mutation level.Meanwhile,GCB has enhanced its association with immune cells in the microenvironment,which plays a positive anti-tumor effect.

Prognostic potential of the small GTPase Ran and its methylation in hepatocellular carcinoma

Background: Hepatocellular carcinoma(HCC) is a common malignant tumor with high mortality. The prognostic significance of Ran, a member of Ras superfamily, remains unclear in HCC patients. Methods: Based on The Cancer Genome Atlas(TCGA) database and Tumor Immune Estimation Resource(TIMER), we analyzed the correlations among Ran expression, promoter methylation and immune cell infiltration. We also investigated the Ran expression levels in HCC tissues and normal tissues by using quantitative real-time PCR. Results: Ran m RNA expression was significantly increased in HCC tissues compared with the normal tissues( P < 0.001). Time-dependent receiver operating characteristic(ROC) curves showed that Ran expression had predictive value of the 1-, 3-and 5-year overall survival for HCC patients, and the areas under the curves(AUC) were 0.747, 0.634 and 0.704, respectively. Cox regression analysis showed that Ran expression was an independent prognostic factor for HCC patients(HR = 1.492, 95% CI: 1.129-1.971, P = 0.005). We also found a negative relationship between Ran m RNA expression and its promoter methylation( r =-0.36, P < 0.001). High Ran expression and promoter hypomethylation predicted worse overall survival and progression-free survival( P < 0.05) and were involved in the progression of HCC. Ran expression exhibited significant correlations with immune infiltrates and prognostic immune-related genes. Conclusions: The present study provides further insight into the prognosis of HCC, and Ran could serve as a biomarker for predicting the survival of HCC patients.

Signature Based on Six Autophagy-related Genes to Predict Prognosis of Head and Neck Squamous Cell Carcinoma

Objective The present study aimed to develop an autophagy-related gene prognostic prediction model to provide survival risk prediction for head and neck squamous cell carcinoma(HNSCC)patients.Methods The K-mean cluster analysis was performed on HNSCC samples based on the expression values of 210 autophagy-related genes for candidate signature gene selection.LASSO Cox regression analysis was generated using the potential genes and the risk score was calculated from the prognosis model.The risk score was processed as an independent prognostic indicator to construct the nomogram model.The immune status including immune cell infiltration ratio and checkpoints of patients with HNSCC in high-and low-risk groups was also explored.Results LASSO Cox regression analysis was performed on the selected autophagy-related genes.According to the lambda value corresponding to the number of different genes in the LASSO Cox analysis,six genes(GABARAPL2,SAR1A,ST13,GAPDH,FADD and LAMP1)were finally chosen.The risk score based on the genes was generated,which was an independent prognostic marker for HNSCC.The prognostic prediction model(nomogram)was further optimized by the independent prognostic factors(risk score),which can better predict the prognosis and survival of patients.With the risk score and prognosis model,eight types of immune cells and six key immune checkpoints(CTLA4,PD1,IDO1,TDO2,LAG3,TIGIT)displayed expression specificity.Conclusion This study identified several potential prognostic biomarkers and established an autophagy-related prognostic prediction model for HNSCC,which provides a valuable reference for future clinical research.

A novel prognostic gene signature,nomogram and immune landscape based on tanshinone IIA drug targets for hepatocellular carcinoma:Comprehensive bioinformatics analysis and in vitro experiments

Tanshinone IIA,one of the main ingredients of Danshen,is used to treat hepatocellular carcinoma(HCC).However,potential targets of the molecule in the therapy of HCC are unknown.Methods:In this study,we collected the tanshinone IIA targets from public databases for investigation.We screened differentially expressed genes(DEGs)across HCC and normal tissues using mRNA expression profiles from The Cancer Genome Atlas(TCGA).Univariate Cox regression analysis and least absolute shrinkage and selection operator(LASSO)Cox regression models were used to identify and construct the prognostic gene signature.Results:Finally,we discovered common genes across tanshinone IIA targets and HCC DEGs.We reported Fatty acid binding protein-6(FABP6),Polo-like Kinase 1(PLK1),deoxythymidylate kinase(DTYMK),Uridine Cytidine Kinase 2(UCK2),Enhancer of Zeste Homolog 2(EZH2),and Cytochrome P4502C9(CYP2C9)as components of a gene signature.The six-gene signature’s prognostic ability was evaluated using the Kaplan-Meier curve,time-dependent receiver operating characteristic(ROC),multivariate Cox regression analysis,and the nomogram.The mRNA level and protein expression of UCK2 were experimentally validated after treatment with different concentrations of tanshinone IIA in HEPG2 cells.CIBERSORTx,TIMER2.0,and GEPIA2 tools were employed to explore the relationship between the prognostic signature and immune cell infiltration.Conclusion:We established a six-gene signature as a reliable model with significant therapeutic possibility for prognosis and overall survival estimation in HCC patients,which might also benefit medical decision-making for appropriate treatment.