Prognostic signature for hepatocellular carcinoma based on 4 pyroptosis-related genes.

BACKGROUND: Hepatocellular carcinoma (HCC) is a cancer with a poor prognosis. Many recent studies have suggested that pyroptosis is important in tumour progression. However, the role of pyroptosis-related genes (PRGs) in HCC remains unclear. MATERIALS AND METHODS: We identified differentially expressed PRGs in tumours versus normal tissues. Through univariate, LASSO, and multivariate Cox regression analyses, a prognostic PRG signature was established. The signature effectiveness was evaluated by time-dependent receiver operating characteristic (t-ROC) curve and Kaplan-Meier (KM) survival analysis. The signature was validated in the ICGC (LIRI-JP) cohort. In addition, single-sample gene enrichment analysis (ssGSEA) showed the infiltration of major immune cell types and the activity of common immune pathways in different subgroups. RESULTS: Twenty-nine pyroptosis-related DEGs from The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset were detected, and four genes (CTSV, CXCL8, MKI67 and PRF1) among them were selected to construct a prognostic signature. Then, the patients were divided into high- and low-risk groups. The pyroptosis-related signature was significantly associated with overall survival (OS). In addition, the patients in the high-risk group had lower levels of immune infiltration. CONCLUSION: The prognostic signature for HCC based on 4 pyroptosis-related genes has reliable prognostic and predictive value for HCC patients.

Tumor-associated macrophages promote human hepatoma Huh-7 cell migration and invasion through the Gli2/IGF-II/ERK1/2 axis by secreting TGF-ß1.

AIM: In this study, we explored the ability of TAMs to affect the malignant phenotype of human hepatoma Huh-7 cells through the Gli2/IGF-II/ERK1/2 pathway. METHODS: The TAMs were characterized by flow cytometry and ELISA assays. Huh-7 cells were treated with conditioned medium of TAMs (TAMs-CM), and the proliferation, migration and invasion abilities were measured by CCK-8, Transwell and scratch assays. The levels of TGF-ß1, Gli2, IGF-II and related proteins in the ERK1/2 pathway and the epithelial-mesenchymal transition (EMT) process were examined by RT-qPCR and western blot. Huh-7 cells were injected subcutaneously into nude mice with TAMs to explore the role of TAMs in tumor growth. RESULTS: The expression levels of TGF-ß1, Gli2 and IGF-II and the cell proliferation, migration and invasion abilities were elevated in Huh-7 cells treated with TAMs-CM. TGF-ß1 was upregulated in the conditioned medium and was found to be involved in the promotion of migration, invasion and the EMT of Huh-7 cells. The activation of TGF-ß1 signaling increased the expression of Gli2. Knockdown of Gli2 decreased the expression of IGF-II and also reversed the promotional effect of the conditioned medium on migration, invasion and the EMT of Huh-7 cells. TGF-ß1/Gli2/IGF-II signaling was shown to promote the malignant phenotype of Huh-7 cells by activating the ERK1/2 signaling pathway. Further, TGF-ß1 knockdown attenuated the influence of TAMs on tumor growth in mouse model. CONCLUSION: The TGF-ß1 secreted by TAMs promotes the migration, invasion and EMT of human hepatoma Huh-7 cells through the Gli2/IGF-II/ERK1/2 pathway.

Clinical and genomic landscape of hepatocellular carcinoma subtypes with various proportions of nonleukocyte stromal cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies and inflicts high mortality worldwide. The effect of tumor microenvironment components on HCC oncogenesis remains unknown. In particular, the nonleukocyte portion of the stromal fraction (SF) is poorly understood. METHODS: We comprehensively evaluated the proportional cell counts and gene expression data from The Cancer Genome Atlas (TCGA) to examine the contributions of cell components to the tumor microenvironment. Single-cell sequencing data from the Gene Expression Omnibus (GEO) were also analyzed to verify the association between the nonleukocyte SF and genes. We classified HCC using a hierarchical clustering method based on diversity of nonleukocyte SF-related gene expression among different components, and we used an appropriate GEO dataset to verify the clusters with a support vector machine (SVM) model. The prognosis of subtypes and their relationship with tumor microenvironmental cell proportions, clinicopathogenesis factors, and other indicators were evaluated. RESULTS: Based on linear regression, 711 genes related to nonleukocyte SF were selected from the TCGA dataset. We classified HCC into three subtypes using genes related to the nonleukocyte SF. Additionally, the GEO single-cell sequencing data confirmed the relationship between genes and the nonleukocyte SF. The tumor microenvironment of Type 2 contained the most significant mutually reinforcing interaction between the nonleukocyte SF and tumor cells. Meanwhile, Type 2 patients had the poorest prognosis and the most severe tumor-node-metastasis (TNM) stages, histological grades, etc. The analysis based on the GEO dataset verified the classification results with an SVM model. Type 2 was associated with worse clinicopathological characteristics, including tumor grading and staging, than the other types. In addition, the pathway analysis revealed that signals related to the SF and cell proliferation were significantly enhanced in Type 2 compared to the other group, which consisted of Types 1 and 3. CONCLUSION: The nonleukocyte SF in the tumor microenvironment contributed greatly to HCC oncogenesis. We can use these HCC classification criteria to stratify patients into subtypes for personalized treatment.

Exploration of the effects of the CYCLOPS gene RBM17 in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most lethal and malignant tumours worldwide. New therapeutic targets for HCC are urgently needed. CYCLOPS (copy number alterations yielding cancer liabilities owing to partial loss) genes have been noted to be associated with cancer-targeted therapies. Therefore, we intended to explore the effects of the CYCLOPS gene RBM17 on HCC oncogenesis to determine if it could be further used for targeted therapy. METHODS: We collected data on 12 types of cancer from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) queries for comparison with adjacent non-tumour tissues. RBM17 expression levels, clinicopathological factors and survival times were analysed. RNAseq data were downloaded from the Encyclopaedia of DNA Elements database for molecular mechanism exploration. Two representative HCC cell models were built to observe the proliferation capacity of HCC cells when RBM17 expression was inhibited by shRBM17. Cell cycle progression and apoptosis were also examined to investigate the pathogenesis of RBM17. RESULTS: Based on 6,136 clinical samples, RBM17 was markedly overexpressed in most cancers, especially HCC. Moreover, data from 442 patients revealed that high RBM17 expression levels were related to a worse prognosis. Overexpression of RBM17 was related to the iCluster1 molecular subgroup, TNM stage, and histologic grade. Pathway analysis of RNAseq data suggested that RBM17 was involved in mitosis. Further investigation revealed that the proliferation rates of HepG2 (P = 0.003) and SMMC-7721 (P = 0.030) cells were significantly reduced when RBM17 was knocked down. In addition, RBM17 knockdown also arrested the progression of the cell cycle, causing cells to halt at the G2/M phase. Increased apoptosis rates were also found in vitro. CONCLUSION: These results suggest that RBM17 is a potential therapeutic target for HCC treatment.

miR-206 inhibits cell proliferation, invasion, and migration by down-regulating PTP1B in hepatocellular carcinoma.

Protein tyrosine phosphatase 1B (PTP1B) has been reported as an oncogene in hepatocellular carcinoma (HCC). However, how PTP1B is regulated in HCC remains unclear. MicroRNAs (miRNAs) are a class of small non-coding RNAs involved many biological processes including tumorigenesis. In this study, we investigated whether miRNA participated in the regulation of PTP1B in HCC. We found that miR-206, which was down-regulated during tumorigenesis, inhibited HCC cell proliferation and invasion. Overexpression of miR-206 inhibited proliferation, invasion, and migration of HCC cell lines HepG2 and Huh7. Mechanistically, we demonstrated that miR-206 directly targeted PTP1B by binding to the 3'-UTR of PTP1B mRNA as demonstrated by the luciferase reporter assay. Overexpression miR-206 inhibited PTP1B expression while miR-206 inhibition enhanced PTP1B expression in HepG2 and Huh7 cells. Functionally, the regulatory effect on cell proliferation/migration/invasion of miR-206 was reversed by PTP1B overexpression. Furthermore, tumor inoculation nude mice model was used to explore the function of miR-206 in vivo Our results showed that overexpression of miR-206 drastically inhibited tumor development. In summary, our data suggest that miR-206 inhibits HCC development by targeting PTP1B.

Alternatively activated NUSAP1 promotes tumor growth and indicates poor prognosis in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies with high mortality. The key genes involved in initiation and development of HCC is not entirely clear. METHODS: We performed a meta-analysis of available transcriptome data from 6 independent HCC datasets [5 datasets from the Gene Expression Omnibus (GEO) and 1 dataset from The Cancer Genome Atlas (TCGA)]. The associations of the nucleolar and spindle-associated protein 1 (NUSAP1) expression level with clinicopathological factors and survival times were analyzed. Two representative HCC cell models were built to observe the proliferation capacity of HCC cells when NUSAP1 expression was inhibited by shNUSAP1. RESULTS: Based on the transcriptome and survival data in the GEO and TCGA databases, NUSAP1 gene was markedly upregulated in HCC. High expression of NUSAP1 in HCC is related to the iCluster1 molecular subgroup, poor survival, poor tumor differentiation and TNM stage. Additionally, pathway analysis based on RNAseq data suggested that NUSAP1 could activate the expression of genes involves in cell proliferation. Furthermore, downregulation of NUSAP1 expression could significantly inhibit the proliferation of SMMC-7721 and Huh7 cells in vitro. CONCLUSIONS: Our study provides evidence that NUSAP1 may serve as a candidate prognostic marker and a target for future therapeutic intervention in HCC.

Interferon-α inhibits cell migration and invasion and induces the expression of antiviral proteins in Huh-7 cells transfected with hepatitis B virus X gene-expressing lentivirus.

Hepatitis B virus (HBV) X protein (HBx) serves an important role in HBV infection and the development of HBV-related liver cancer. Interferon-α (IFN-α) is used to treat patients with HBV; however, the role of IFN-α in the development of HBV-related liver cancer remains unclear. The present study established a new HBV-related liver cancer model (Huh-7-HBx) by transfecting the hepatoma cell line Huh-7, with HBx-expressing lentivirus. Following IFN-α treatment, cell viability, migration and invasion, as well as the expression of antiviral proteins in Huh-7-HBx, were subsequently determined. The results demonstrated that HBx-expressing lentivirus had no significant effect on cell viability but promoted the migration and invasion of Huh-7 cells. The expression of the antiviral genes IFN α and ß receptor subunit 1 (IFNAR1), IFNAR2, IFN-stimulated gene factor 3, double-stranded RNA-activated protein kinase and ribonuclease L, was also increased. Following treatment of Huh-7-HBx cells with IFN-α, the expression of antiviral genes was increased at the level of transcription and translation, whereas cell migration and invasion was decreased. The present study suggests that IFN-α may attenuate the development of HBV-related liver cancer by reducing cell migration and invasion and promoting the expression of antiviral proteins.

SAMMSON drives the self-renewal of liver tumor initiating cells through EZH2-dependent Wnt/ß-catenin activation.

Liver cancer is one of the most serious cancers all over the world. Liver tumor initiating cells (TICs) account for tumor initiation and metastasis. However, the regulatory mechanism of liver TICs remains unclear. Here we found long noncoding RNA SAMMSON is highly expressed in liver cancer and liver TICs. SAMMSON silenced cells show impaired self-renewal capacity, while, its overexpression induces enhanced self-renewal. SAMMSON drives the activation of Wnt/ß-catenin signaling, and thus promotes liver TIC self-renewal. SAMMSON interacts with EZH2, a core component of PRC2 complex, and inhibits the expression of CTNNBIP1 through EZH2 dependent manner. SAMMSON binds to CTNNBIP1 promoter and recruits EZH2 to CTNNBIP1 promoter. What's more, targeting liver TICs through SAMMSON, EZH2 and Wnt/ß-catenin signaling impaired liver TIC self-renewal, decreased tumor propagation and severity. Taken together, SAMMSON drives liver TIC self-renewal through EZH2-dependent Wnt/ß-catenin activation.

Role of growth factor receptor-bound 2 in CCl4-induced hepatic fibrosis.

BACKGROUND: Growth Factor Receptor-bound 2 (GRB2) plays a crucial role in regulation of cellular function including proliferation and differentiation, and we previously identified GRB2 as promoting HSCs (HSCs) proliferation. However, the underlying mechanisms that are involving in the regulation of GRB2 in hepatic fibrogenesis remain unknown. METHODS: In the present study, we tested the function of GRB2 in hepatic fibrosis. Hepatic fibrosis was induced by subcutaneous CCl4 administration at a dose of 3mL/kg in rats. The rat HSC cell line HSC-T6 were cultured for proliferation investigation by CCK-8 and BrdU incorporation method. The levels of GRB2, HMGB1, PI3K/AKT, COL1A1 and α-SMA were analyzed by western blot or real-time PCR. RESULTS: showed that the expression of GRB2 and HMGB1 was obviously increased in liver tissues of hepatic fibrosis rats accompanied by up-regulation of COL1A1 and α-SMA. In cultured HSCs, application of exogenous HMGB1 induced cell proliferation and cell proliferation rate concomitantly with up-regulation of GRB2 expression and PI3K/AKT phosphorylation. The effects of HMGB1-induced proliferation of HSCs and up-regulation of COL1A1 and α-SMA were abolished by GRB2 siRNA. HMGB1-induced proliferation of HSCs and up-regulation of COL1A1 and α-SMA was reversed in the presence of LY294002, an inhibitor of PI3K inhibitor. CONCLUSIONS: These findings suggest that GRB2 plays an important role in CCl4-induced hepatic fibrosis by regulating HSCs' function, and up-regulation of GRB2 induced by HMGB1 is mediated via the PI3K/AKT pathway.