Single-Cell RNA Sequencing Reveals the Role of Phosphorylation-Related Genes in Hepatocellular Carcinoma Stem Cells.
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ABSTRACT:
SUBMITTER: Yao F
PROVIDER: S-EPMC8763978 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundSingle-cell RNA sequencing, also known as scRNA-seq, is a method profiling cell populations on an individual cell basis. It is particularly useful for more deeply understanding cell behavior in a complicated tumor microenvironment. Although several previous studies have examined scRNA-seq for hepatocellular carcinoma (HCC) tissues, no one has tested and analyzed HCC with different stages.MethodsIn this investigation, immune cells isolated from surrounding normal tissues and cancer tissues from 3 II-stage and 4 III-stage HCC cases were subjected to deep scRNA-seq. The analysis included 15 samples. We distinguished developmentally relevant trajectories, unique immune cell subtypes, and enriched pathways regarding differential genes. Western blot and co-immunoprecipitation were performed to demonstrate the interaction between fatty acid binding protein 1 (FABP1) and peroxisome proliferator-activated receptor gamma(PPARG). In vivo experiments were performed in a C57BL/6 mouse model of HCC established via subcutaneous injection.ResultsFABP1 was discovered to be overexpressed in tumor-associated macrophages (TAMs) with III-stage HCC tissues compared with II-stage HCC tissues. This finding was fully supported by immunofluorescence detection in significant amounts of HCC human samples. FABP1 deficiency in TAMs inhibited HCC progression in vitro. Mechanistically, FABP1 interacted with PPARG/CD36 in TAMs to increase fatty acid oxidation in HCC. When compared with C57BL/6 mice of the wild type, tumors in FABP1-/- mice consistently showed attenuation. The FABP1-/- group's relative proportion of regulatory T cells and natural killer cells showed a downward trend, while dendritic cells, M1 macrophages, and B cells showed an upward trend, according to the results of mass cytometry. In further clinical translation, we found that orlistat significantly inhibited FABP1 activity, while the combination of anti-programmed cell death 1(PD-1) could synergistically treat HCC progression. Liposomes loaded with orlistat and connected with IR780 probe could further enhance the therapeutic effect of orlistat and visualize drug metabolism in vivo.ConclusionsScRNA-seq atlas revealed an FABP1-dependent immunosuppressive environment in HCC. Orlistat significantly inhibited FABP1 activity, while the combination of anti-PD-1 could synergistically treat HCC progression. This study identified new treatment targets and strategies for HCC progression, contributing to patients with advanced HCC from new perspectives.
| S-EPMC10679975 | biostudies-literature
Project description:Background: Hepatocellular carcinoma (HCC) is among the deadliest cancers worldwide, and advanced HCC is difficult to treat. Identifying specific cell subpopulations in the tumor microenvironment and exploring interactions between the cells and their environment are crucial for understanding the development, prognosis, and treatment of tumors. Methods: In this study, we constructed a tumor ecological landscape of 14 patients with HCC from 43 tumor tissue samples and 14 adjacent control samples. We used bioinformatics analysis to reveal cell subpopulations with potentially specific functions in the tumor microenvironment and to explore the interactions between tumor cells and the tumor microenvironment. Results: Immune cell infiltration was evident in the tumor tissues, and BTG1 + RGS1 + central memory T cells (Tcms) interact with tumor cells through CCL5-SDC4/1 axis. HSPA1B may be associated with remodeling of the tumor ecological niche in HCC. Cancer-associated fibroblasts (CAFs) and macrophages (TAMs) were closely associated with tumor cells. APOC1 + SPP1 + TAM secretes SPP1, which binds to ITGF1 secreted by CAFs to remodel the tumor microenvironment. More interestingly, FAP + CAF interacts with naïve T cells via the CXCL12-CXCR4 axis, which may lead to resistance to immune checkpoint inhibitor therapy. Conclusion: Our study suggests the presence of tumor cells with drug-resistant potential in the HCC microenvironment. Among non-tumor cells, high NDUFA4L2 expression in fibroblasts may promote tumor progression, while high HSPA1B expression in central memory T cells may exert anti-tumor effects. In addition, the CCL5-SDC4/1 interaction between BTG1 + RGS1 + Tcms and tumor cells may promote tumor progression. Focusing on the roles of CAFs and TAMs, which are closely related to tumor cells, in tumors would be beneficial to the progress of systemic therapy research.
| S-EPMC10272598 | biostudies-literature
Project description:Efficacy of immunotherapy in hepatocellular carcinoma (HCC) is blocked by its high degree of inter- and intra-tumor heterogeneity and immunosuppressive tumor microenvironment. However, the correlation between tumor heterogeneity and immunosuppressive microenvironment in HCC has not been well addressed. Here, we endeavored to dissect inter- and intra-tumor heterogeneity in HCC and uncover how they contribute to the immunosuppressive microenvironment. We performed consensus molecular subtyping with non-negative matrix factorization (NMF) clustering to stratify the inter-heterogeneity profile of HCC tumors. We grouped HCC tumors from the Cancer Genome Atlas (TCGA) patients into three subtypes (S1, S2 and S3), where S1 was characterized as a 'hot tumor' profile with high expression level of T cell genes and rate of immune scores. S2 was characterized as a 'cold tumor' profile with the highest tumor purity score, and S3 as an 'immunosuppressed tumor' profile with the poorest prognosis and a high expression level of immunosuppressive genes such as cytotoxic T-lymphocyte-associated protein-4, TIGIT, and PDCD1. Moreover, we combined weighted gene co-expression network analysis and single-cell regulatory network inference and clustering (SCENIC) in the single-cell dataset of the S3-like subtype (CS3) and identified a transcription factor, BATF, which could upregulate immunosuppressive genes. Finally, we identified a cell interaction network in which a myeloid-derived suppressor cell-like macrophage subtype could promote the formation of immunosuppressive T-cells.
| S-EPMC9168757 | biostudies-literature
Project description:BackgroundHepatocellular carcinoma (HCC) is the third leading cause of death in the world, characterized by high morbidity, poor prognosis and high mortality. Histone modifications regulate intracellular gene expression at the post-transcriptional level, and disturbances in the regulatory pattern of histone modifications at individual locus or across the genome can lead to tumorigenesis of HCC. In this study, we constructed a prognosis-related histone phosphorylation regulated (HPR) genes signature and elucidated whether HPR genes can predict overall survival in HCC patients.MethodsDifferentially expressed genes were screened using TCGA, ICGC and GEO databases, and a new risk signature was constructed by univariate Cox regression and Lasso regression analysis. Predictive nomograms were established by multivariate Cox regression of risk scores and clinical parameters, calibration curve and decision curve analysis were used to evaluate the models. The ssGSEA methods were used to determine the effect of risk scores on the tumor immune microenvironment. Data for HCC single-cell RNA sequencing (scRNA-seq) have been downloaded from Gene Expression Omnibus (GEO) to understand the role of HPR genes in tumorigenesis.ResultsOur analyses of nine HPR genes provided prognostic insights. Overall survival in the low-risk and high-risk groups was statistically higher, respectively (P<0.001). Cox regression analysis revealed that the risk score is a significant predictor of HCC outcomes (HR=2. 2.62, 95%CI: 1.248-5.514, P=0.011). In addition, a nomogram combining risk scores with TNM stages was constructed and tested from calibration curves and decision curves (AUC=0.780). MHC-class-I genes, iDCs, Macrophages, Tfh, Treg, Th2 were overexpressed in the high-risk group.ConclusionHPR genes risk score is closely related to the prognosis of HCC, tumor immune process and tumor cell progression.
| S-EPMC9470838 | biostudies-literature
Project description:Intratumor molecular heterogeneity of hepatocellular carcinoma is partly attributed to the presence of hepatic cancer stem cells (CSCs). Different CSC populations defined by various cell surface markers may contain different oncogenic drivers, posing a challenge in defining molecularly targeted therapeutics. We combined transcriptomic and functional analyses of hepatocellular carcinoma cells at the single-cell level to assess the degree of CSC heterogeneity. We provide evidence that hepatic CSCs at the single-cell level are phenotypically, functionally, and transcriptomically heterogeneous. We found that different CSC subpopulations contain distinct molecular signatures. Interestingly, distinct genes within different CSC subpopulations are independently associated with hepatocellular carcinoma prognosis, suggesting that a diverse hepatic CSC transcriptome affects intratumor heterogeneity and tumor progression. CONCLUSION:Our work provides unique perspectives into the biodiversity of CSC subpopulations, whose molecular heterogeneity further highlights their role in tumor heterogeneity, prognosis, and hepatic CSC therapy. (Hepatology 2018;68:127-140).
| S-EPMC6033650 | biostudies-literature
Project description:Dysregulation of expression of functional genes and pathways plays critical roles in the etiology and progression of hepatocellular carcinoma (HCC). Next generation-based RNA sequencing (RNA-seq) offers unparalleled power to comprehensively characterize HCC at the whole transcriptome level. In this study, 17 fresh-frozen HCC samples with paired non-neoplastic liver tissue from Caucasian patients undergoing liver resection or transplantation were used for RNA-seq analysis. Pairwise differential expression analysis of the RNA-seq data was performed to identify genes, pathways, and functional terms differentially regulated in HCC versus normal tissues. At a false discovery rate (FDR) of 0.10, 13% (n = 4335) of transcripts were up-regulated and 19% (n = 6454) of transcripts were down-regulated in HCC versus non-neoplastic tissue. Eighty-five Kyoto Encyclopedia of Genes and Genomes pathways were differentially regulated (FDR, <0.10), with almost all pathways (n = 83) being up-regulated in HCC versus non-neoplastic tissue. Among the top up-regulated pathways was oxidative phosphorylation (hsa00190; FDR, 1.12E-15), which was confirmed by Database for Annotation, Visualization, and Integrated Discovery (DAVID) gene set enrichment analysis. Consistent with potential oxidative stress due to activated oxidative phosphorylation, DNA damage-related signals (e.g., the up-regulated hsa03420 nucleotide excision repair [FDR, 1.14E-04] and hsa03410 base excision repair [FDR, 2.71E-04] pathways) were observed. Among down-regulated genes (FDR, <0.10), functional terms related to cellular structures (e.g., cell membrane [FDR, 3.05E-21] and cell junction [FDR, 2.41E-07], were highly enriched, suggesting compromised formation of cellular structure in HCC at the transcriptome level. Interestingly, the olfactory transduction (hsa04740; FDR, 1.53E-07) pathway was observed to be down-regulated in HCC versus non-neoplastic tissue, suggesting impaired liver chemosensory functions in HCC. Our findings suggest oxidative phosphorylation and the associated DNA damage may be the major driving pathologic feature in HCC.
| S-EPMC9315135 | biostudies-literature
Project description:IntroductionThe conflict between cancer cells and the host immune system shapes the immune tumour microenvironment (TME) in hepatocellular carcinoma (HCC). A deep understanding of the heterogeneity and intercellular communication network in the TME of HCC will provide promising strategies to orchestrate the immune system to target and eradicate cancers.MethodsHere, we performed single-cell RNA sequencing (scRNA-seq) and computational analysis of 35786 unselected single cells from 3 human HCC tumour and 3 matched adjacent samples to elucidate the heterogeneity and intercellular communication network of the TME. The specific lysis of HCC cell lines was examined in vitro using cytotoxicity assays. Granzyme B concentration in supernatants of cytotoxicity assays was measured by ELISA.ResultsWe found that VCAN+ tumour-associated macrophages (TAMs) might undergo M2-like polarization and differentiate in the tumour region. Regulatory dendritic cells (DCs) exhibited immune regulatory and tolerogenic phenotypes in the TME. Furthermore, we observed intensive potential intercellular crosstalk among C1QC+ TAMs, regulatory DCs, regulator T (Treg) cells, and exhausted CD8+ T cells that fostered an immunosuppressive niche in the HCC TME. Moreover, we identified that the TIGIT-PVR/PVRL2 axis provides a prominent coinhibitory signal in the immunosuppressive TME. In vitro, antibody blockade of PVR or PVRL2 on HCC cell lines or TIGIT blockade on immune cells increased immune cell-mediated lysis of tumour cell. This enhanced immune response is paralleled by the increased secretion of Granzyme B by immune cells.DiscussionCollectively, our study revealed the functional state, clinical significance, and intercellular communication of immunosuppressive cells in HCC at single-cell resolution. Moreover, PVR/PVRL2, interact with TIGIT act as prominent coinhibitory signals and might represent a promising, efficacious immunotherapy strategy in HCC.
| S-EPMC10293927 | biostudies-literature
Project description:BackgroundLiver cirrhosis is a well-known risk factor for hepatocellular carcinoma (HCC). However, some HCC cases can also originate from non-cirrhotic livers. The aim of this study was to identify key circular RNAs (circRNAs) associated with the tumorigenesis of non-cirrhotic liver disease.MethodsThe differently expressed circRNAs between non-cirrhotic and cirrhotic HCCs were assessed with use of high-throughput circRNAs sequencing and validated with quantitative reverse transcription polymerase chain reaction (qRT-PCR). Potential biological functions of these dysregulated circRNAs were predicted with use of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. A circRNA-miRNA-mRNA regulation network was constructed as achieved with use of miRanda software and visualized using Cytoscape software. Biological functions of the four most prominent dysregulated circRNAs identified were confirmed by in vitro experiments. Moreover, possible translations of these dysregulated circRNAs were also predicted.ResultsA total of 393 dysregulated circRNAs were identified between non-cirrhotic and cirrhotic HCC, including 213 that were significantly up-regulated and 180 significantly down-regulated circRNAs. Expression levels of the six most prominent dysregulated circRNAs were further validated using qRT-PCR. Many tumor related miRNAs were involved in the circRNA-miRNA-mRNA networks, including miR-182-5p, miR-561-3p, miR-125a-5p, miR-145, miR-23b-3p and miR-30e-3p, and downstream mRNAs of dysregulated circRNAs were significantly related with biological processes involved in the progression of tumors, including proliferation, migration, differentiation, and focal adhesion. Results from the in vitro experiments demonstrated that the most prominent dysregulated circRNAs exerted notable effects upon the proliferation and migration of HCC cells. Finally, we also identified 19 dysregulated circRNAs having potential for the coding of functional peptides.ConclusionThe results of this present study indicate that circRNAs may play important roles in tumorigenesis of non-cirrhotic HCC. Such findings provide some novel insights and pave the way for the development of future studies directed at investigating the initiation and treatment of HCC.
| S-EPMC9356431 | biostudies-literature
Project description:Background: Natural killer (NK) cells are a type of innate immune cell that recognize and eliminate tumor cells and infected cells, without prior sensitization or activation. Herein, we aimed to construct a predictive model based on NK cell-related genes for hepatocellular carcinoma (HCC) patients and assess the feasibility of utilizing this model for prognosis prediction. Methods: Single-cell RNA-seq data were obtained from the Gene Expression Omnibus (GEO) database to identify marker genes of NK cells. Univariate Cox and lasso regression were performed to further establish a signature in the TCGA dataset. Subsequently, qPCR and immunohistochemistry (IHC) staining were employed to validate the expression levels of prognosis signature genes in HCC. The effectiveness of the model was further validated using two external cohorts from the GEO and ICGC datasets. Clinical characteristics, prognosis, tumor mutation burden, immune microenvironments, and biological function were compared for different genetic subtypes and risk groups. Finally, molecular docking was performed to evaluate the binding affinity between the hub gene and chemotherapeutic drugs. Results: A total of 161 HCC-related NK cell marker genes (NKMGs) were identified, 28 of which were significantly associated with overall survival in HCC patients. Based on differences in gene expression characteristics, HCC patients were classified into three subtypes. Ten prognosis genes (KLRB1, CD7, LDB2, FCER1G, PFN1, FYN, ACTG1, PABPC1, CALM1, and RPS8) were screened to develop a prognosis model. The model not only demonstrated excellent predictive performance on the training dataset, but also were successfully validated on two independent external datasets. The risk scores derived from the model were shown to be an independent prognosis factor for HCC and were correlated with pathological severity. Moreover, qPCR and IHC staining confirmed that the expression of the prognosis genes was generally consistent with the results of the bioinformatic analysis. Finally, molecular docking revealed favorable binding energies between the hub gene ACTG1 and chemotherapeutic drugs. Conclusion: In this study, we developed a model for predicting the prognosis of HCC based on NK cells. The utilization of NKMGs as innovative biomarkers showed promise in the prognosis assessment of HCC.
| S-EPMC10307919 | biostudies-literature
Project description:The long reads of Nanopore sequencing permit accurate transcript assembly and ease in discovering novel transcripts with potentially important functions in cancers. The wide adoption of Nanopore sequencing for transcript quantification, however, is largely limited by high costs. To address this issue, we developed a bioinformatics software, NovelQuant, that can specifically quantify long-read-assembled novel transcripts with short-read sequencing data. Nanopore Direct RNA Sequencing was carried out on three hepatocellular carcinoma (HCC) patients' tumor, matched portal vein tumor thrombus, and peritumor to reconstruct the HCC transcriptome. Then, based on the reconstructed transcriptome, NovelQuant was applied on Illumina RNA sequencing data of 59 HCC patients' tumor and paired peritumor to quantify novel transcripts. Our further analysis revealed 361 novel transcripts dysregulated in HCC and that 101 of them were significantly associated with prognosis. There were 19 novel prognostic transcripts predicted to be long noncoding RNAs (lncRNAs), and some of them had regulatory targets that were reported to be associated with HCC. Additionally, 42 novel prognostic transcripts were predicted to be protein-coding mRNAs, and many of them could be involved in xenobiotic metabolism. Moreover, the tumor-suppressive roles of two representative novel prognostic transcripts, CDO1-novel (lncRNA) and CYP2A6-novel (protein-coding mRNA), were further functionally validated during HCC progression. Overall, the current study shows a possibility of combining long- and short-read sequencing to explore functionally important novel transcripts in HCC with accuracy and cost-efficiency, which expands the pool of molecular biomarkers that could enhance our understanding of the molecular mechanisms of HCC.
| S-EPMC8409408 | biostudies-literature