Overexpression of SSR2 promotes proliferation of liver cancer cells and predicts prognosis of patients with hepatocellular carcinoma

Signal Sequence Receptor Subunit 2 (SSR2) is a key endoplasmic reticulum gene involved in protein folding and processing. Previous studies found that it was upregulated in several cancers, but its precise role in hepatocellular carcinoma (HCC) remains unclear. To have a better understanding of this gene in HCC, we examined the expression of SSR2 in HCC tissues by analysing The Cancer Genome Atlas (TCGA) data and immunohistochemistry. We also assessed the association between SSR2 expression and clinicopathological characteristics of HCC patients and patient survival. Potential function of SSR2 was predicted through GSEA and protein–protein interaction analysis. MTT, flowcytometry, transwell and a nude mice xenograft model were employed to investigate the biological functions in vivo and in vitro. The results showed that the expression of SSR2 was significantly increased in HCC tissues, and SSR2 expression was associated with several clinical characteristics. In addition, patients with higher SSR2 expression had poorer survival. Enrichment analysis suggested that SSR2 was probably involved in biological process or signalling pathways related to G2/M checkpoint, passive transmembrane transporter activity, ATF2_S_UP. V1_UP and ncRNA metabolic process. Further experimental study showed that SSR2 knockdown inhibited cell proliferation, migration and invasion ability and promoted apoptosis and cell cycle arrest in vitro. Moreover, downregulation of SSR2 also repressed the growth of HepG2 cells in vivo. In conclusion, our study suggests that SSR2 may act as an oncogene in HCC.     

Upregulation of cystatin SN promotes hepatocellular carcinoma progression and predicts a poor prognosis

Cystatin SN, a specific cysteine protease inhibitor, is thought to be involved in various malignant tumors. Therefore, we evaluated the role of cystatin SN in hepatocellular carcinoma (HCC). Notably, cystatin SN was elevated in tumorous samples and cells. Moreover, overexpression of cystatin SN was correlated with tumor diameter and TNM stage. Cox multivariate analysis displayed that cystatin SN was an independent prognosis indicator and that high cystatin SN level was associated with a dismal prognosis. Moreover, cystatin SN enhancement facilitated the proliferation, migratory, and invasive potential of Huh7 and HCCLM3 cells, whereas cystatin SN knockdown caused the opposite effect. Cystatin SN also modulated the epithelial-mesenchymal transition progression through the PI3K/AKT pathway. In vivo cystatin SN promoted HCCLM3 cell growth and metastasis in xenograft mice model. Thus, cystatin SN was involved in HCC progression and could be a latent target for HCC treatment.     

CEP131 indicates poor prognosis and promotes cell proliferation and migration in hepatocellular carcinoma

Centrosomal proteins have been implicated in the progression of human diseases. CEP131 plays important roles in centrosome duplication and genome stability, but its role in cancers remains largely unknown. Here, we showed that CEP131 expression was increased in hepatocellular carcinoma (HCC), compared to the paracarcinoma tissues, at both mRNA and protein levels. High CEP131 expression was closely associated with tumor size (P = 0.020), tumor capsule (P = 0.043), TNM stage (P = 0.007) and tumor differentiation (P = 0.019). Furthermore, patients with high expression of CEP131 were accompanied with worse overall and disease-free survivals in our and TCGA cohorts consisting of a total of 802 cases. The prognostic value of CEP131 was further confirmed by stratified survival analysis. Multivariate cox regression model indicated that CEP131 was an independent factor for overall survival (hazard ratio = 1.762, 95% confident interval: 1.443–2.151, P < 0.001). In vitro data demonstrated that nucleophosmin (NPM) physically bound to CEP131 and maintained its protein stability. Overexpression of CEP131 in HCC cell lines enhanced cell proliferation and migration, whereas the knockdown of CEP131 led to the opposite phenotypes. Further studies demonstrated that CEP131 exhibited oncogenic activity via activation of PI3K/AKT signaling pathway. Taken together, our findings suggest CEP131 serves as a potential prognostic biomarker in HCC, and functions as an oncogene in this deadly disease.     

Inhibition of cell proliferation and induction of apoptosis by genistein in experimental hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the leading cause of cancer related deaths in the world, with increasing incidence in many developed countries. Epidemiological data suggest that consumption of soy products may be associated with a decreased risk of cancer. We investigate the effects of genistein on cell proliferation, apoptosis and caspase-3 in DEN induced (200 mg/kg body weight; by single intraperitoneal injection) and Phenobarbital promoted (0.05% through drinking water for 14 successive weeks) cancer-bearing rats. Immunohistochemistry was employed to detect cell proliferating markers proliferating cell nuclear antigen (PCNA), DNA fragmentation was determined by agarose gel electrophoresis and terminal deoxynucleatide transferase dUTP nick labeling (TUNEL) staining and caspase by enzyme-linked immunosorbent assay. We found inhibition of cell proliferation, induction of apoptosis and activation of caspase-3 in genistein treated animals. From these results, we conclude that genistein inhibit cell proliferation, induced apoptosis. This activation of caspsase-3 in genistein treated liver cancer bearing animals correlated well with its apoptosis inducing effect.     

LncRNA OIP5-AS1 predicts poor prognosis and regulates cell proliferation and apoptosis in bladder cancer

Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) is a long intergenic noncoding RNA, which has been suggested to be dysregulated in human cancers and served as tumor suppressor or promoter depending on tumor types. However, the role of OIP5-AS1 in bladder cancer was still unknown. In our study, OIP5-AS1 was overexpressed in bladder cancer, and associated with clinical progression and short overall survival. The loss-of-function studies suggested downregulation of OIP5-AS1 expression decreased cell viability, induced cell-cycle arrest and promoted cell apoptosis in bladder cancer. There was a positive association between OIP5-AS1 expression and OIP5 expression in bladder cancer tissues. Moreover, downregulation of OIP5-AS1 expression reduced messenger RNA and protein levels of OIP5 in bladder cancer cell lines. In conclusion, OIP5-AS1 is a useful biomarker for predicting clinical progression and poor prognosis and promotes cell proliferation through modulating OIP5 expression.     

人肝细胞癌预后不良相关基因的生物信息学分析及其临床意义

目的:筛选肝细胞癌(HCC)预后不良相关基因,并探讨其临床意义。方法:在基因表达综合数据库(GEO)中获取符合分析条件的肝细胞癌全基因组表达谱数据并分析得到差异表达基因(DEGs),再运用生物学信息注释及可视化数据库 (DAVID) 和蛋白相互作用数据库 (String) 分别进行功能富集分析和蛋白质互作用网络的构建。利用癌症基因组图谱数据库(TCGA)和Cox比例风险回归模型对相关差异基因进行预后分析。结果:找到一个符合条件的人类HCC数据库 (GSE84402),共筛选出1141个差异表达基因(DEGs),其中上调基因720个,下调基因421个。基因功能富集分析和蛋白质互作用分析结果显示CDK1、CDC6、CCNA2、CHEK1、CENPE 、PIK3R1、RACGAP1、BIRC5、KIF11和CYP2B6为HCC预后的关键基因。TCGA数据库和Cox回归模型分析显示CDC6、PIK3R1、RACGAP1和KIF11的表达升高,CENPE的表达降低与HCC预后不良密切相关。结论:CDC6、CENPE、PIK3R1、RACGAP1和KIF11可能和HCC的预后不良相关,可作为未来HCC预后研究的参考标志物。     

Musashi2 predicts poor prognosis and invasion in hepatocellular carcinoma by driving epithelial–mesenchymal transition

The high incidence of recurrence and the poor prognosis of hepatocellular carcinoma (HCC) necessitate the discovery of new predictive markers of HCC invasion and prognosis. In this study, we evaluated the expression pattern of two members of a novel oncogene family, Musashi1 (MSI1) and Musashi2 (MSI2) in 40 normal hepatic tissue specimens, 149 HCC specimens and their adjacent non‐tumourous tissues. We observed that MSI1 and MSI2 were significantly up‐regulated in HCC tissues. High expression levels of MSI1 and MSI2 were detectable in 37.6% (56/149) and 49.0% (73/149) of the HCC specimens, respectively, but were rarely detected in adjacent non‐tumourous tissues and were never detected in normal hepatic tissue specimens. Nevertheless, only high expression of MSI2 correlated with poor prognosis. In addition, MSI2 up‐regulation correlated with clinicopathological parameters representative of highly invasive HCC. Further study indicated that MSI2 might enhance invasion of HCC by inducing epithelial–mesenchymal transition (EMT). Knockdown of MSI2 significantly decreased the invasion of HCC cells and changed the expression pattern of EMT markers. Moreover, immunohistochemistry assays of 149 HCC tissue specimens further confirmed this correlation. Taken together, the results of our study demonstrated that MSI2 correlates with EMT and has the potential to be a new predictive biomarker of HCC prognosis and invasion to help guide diagnosis and treatment of post‐operative HCC patients.     

Identification of m6A methyltransferase-related lncRNA signature for predicting immunotherapy and prognosis in patients with hepatocellular carcinoma

N6-methyladenosine (m6A) methyltransferase has been shown to be an oncogene in a variety of cancers. Nevertheless, the relationship between the long non-coding RNAs (lncRNAs) and hepatocellular carcinoma (HCC) remains elusive. We integrated the gene expression data of 371 HCC and 50 normal tissues from The Cancer Genome Atlas (TCGA) database. Differentially expressed protein-coding genes (DE-PCGs)/lncRNAs (DE-lncRs) analysis and univariate regression and Kaplan–Meier (K–M) analysis were performed to identify m6A methyltransferase-related lncRNAs. Three prognostic lncRNAs were selected by univariate and LASSO Cox regression analyses to construct the m6A methyltransferase-related lncRNA signature. Multivariate Cox regression analyses illustrated that this signature was an independent prognostic factor for overall survival (OS) prediction. The Gene Set Enrichment Analysis (GSEA) suggested that the m6A methyltransferase-related lncRNAs were involved in the immune-related biological processes (BPs) and pathways. Besides, we discovered that the lncRNAs signature was correlated with the tumor microenvironment (TME) and the expression of critical immune checkpoints. Tumor Immune Dysfunction and Exclusion (TIDE) analysis revealed that the lncRNAs could predict the clinical response to immunotherapy. Our study had originated a prognostic signature for HCC based on the potential prognostic m6A methyltransferase-related lncRNAs. The present study had deepened the understanding of the TME status of HCC patients and laid a theoretical foundation for the choice of immunotherapy.     

Overexpression of VCC-1 gene in human hepatocellular carcinoma cells promotes cell proliferation and invasion

Vascular endothelial growth factor-correlated chemokine 1 （VCC-1）, a novel chemokine, is hypothesized to be associated with carcinogenesis. VCC-1 is expressed in hepatocellular carcinoma （HCC） cells, but its func- tion remains unknown. To investigate the molecular effects of VCC-1 on HCC cells, the HCC cell line SMMC7721 was stably transfected with the recombinant plasmid pcDNA3.1/VCC-1. Our data demonstrated that overexpression of VCC-1 in SMMC7721 cells significantly enhanced the cellular proliferation, invasive ability, and tumor growth, when compared with both empty vector control cells and parental cells. These results strongly suggest that VCC-1 plays an important role in SMMC7721 invasion and tumor growth, and indicate that VCC-1 may serve as a potential biomarker for anti-HCC therapies.     

Down-regulation of RBP4 indicates a poor prognosis and correlates with immune cell infiltration in hepatocellular carcinoma

Recent research has indicated that metabolically related genes play crucial roles in the pathogenesis of hepatocellular carcinoma (HCC). We evaluated the associations between novel biomarkers and retinol-binding protein 4 (RBP4) for predicting clinical HCC outcomes, hub-related genes, pathway regulation, and immune cells infiltration. Bioinformatic analyses based on data from The Cancer Genome Atlas were performed using online analysis tools. RBP4 expression was low in HCC and was also down-regulated in pan-cancers compared with normal tissues. RBP4 expression was also significantly different based on age (41–60 years old versus 61–80 years old), and low RBP4 expression levels were associated with advanced tumor stages and grades. Higher RBP4 expression was associated with better overall survival time in HCC patients, and we identified a deletion-mutation rate of 1.4% in RBP4. We also identified ten co-expressed genes most related to RBP4 and explored the relationships between six hub genes (APOB, FGA, FGG, SERPINC1, APOA1, and F2) involved in RBP4 regulation. A pathway enrichment analysis for RBP4 indicated complement and coagulation cascades, metabolic pathways, antibiotic biosynthesis pathways, peroxisome proliferator-activated receptor signaling pathways, and pyruvate metabolism pathways. These results suggest that RBP4 may be a novel biomarker for HCC prognosis, and an indicator of low immune response to the disease.     

Transmembrane protein 106C promotes the development of hepatocellular carcinoma

Several protein-coding genes have been identified to play essential roles in cancer biology, and they are dysregulated in many tumors. Transmembrane protein 106C (TMEM106C) is differentially expressed in several human and porcine diseases; however, the expression and biological functions of TMEM106C in hepatocellular carcinoma (HCC) are not clear. In our study, we obtained paired tissue samples from patients undergoing resection for HCC and public databases, which were analyzed for TMEM106C expression using quantitative real-time polymerase chain reaction (qRT-PCR). We further conducted in vitro and in vivo experiments in HCC cell lines and nude mice, respectively, in which TMEM106C was overexpressed or knocked down. Cell-Counting Kit-8 and colony formation experiments were used to determine the influence of TMEM106C on cell proliferation, flow cytometric assays were used to detect the influence on cell cycle distribution and apoptosis, and transwell assays were used for detecting changes in cell migration and invasion. TMEM106C levels were significantly elevated in HCC tissues and cell lines from public databases and our collected specimens from patients. Moreover, higher TMEM106C expression levels predicted a poor prognosis in HCC patients in survival analysis. Overexpression of TMEM106C in HCC cells accelerated cell growth, migration, and invasion, but it inhibited cell apoptosis by targeting forkhead box O-1 (FOXO1) and FOXO3. Conversely, TMEM106C knockdown impeded cell proliferation and metastasis, whereas it enhanced the rate of apoptosis. More important, knockdown of the expression of TMEM106C in HCC cells inhibited the growth of xenograft tumors in vivo. Collectively, these results suggest that TMEM106C acts as an oncogene and can serve as a potential therapeutic target for HCC in the future.     

Elevated expression of circular RNA circ_0008450 predicts dismal prognosis in hepatocellular carcinoma and regulates cell proliferation,apoptosis, and invasion via sponging miR-548p

Hepatocellular carcinoma (HCC) is one of the most frequent malignancies and a main cause of global cancer mortality. In the past decade, circular RNAs (circRNAs) have been proved to play key roles in various cancers. Previously, circ_0008450 was identified upregulated in HCC tissues by high-throughput circRNA sequencing. In this study, quantitative real-time polymerase chain reaction was used to evaluate the expression level of circ_0008450 in human HCC tumor and corresponding nontumor tissue samples, and the association between circ_0008450 expression and clinicopathologic features of patients with HCC was also analyzed. After that, the functions of circ_0008450 in biological behaviors of HCC cells were determined by cell counting kit-8, colony formation, flow cytometry, and the transwell assays. The mechanism of circ_0008450 was explored by the bioinformatic analysis and dual-luciferase reporter assay. The expression of circ_0008450 is upregulated in HCC tissue specimens and cell lines. Patients with a high circ_0008450 expression usually bear a lower 5-year survival rate. Silencing of circ_0008450 in Huh-7 cells inhibited cell viability, migration, and invasion, whereas cell apoptosis was increased. Conversely, its overexpression in HepG2 cells leads to absolutely inverse results. In addition, circ_0008450 was proved to be a sponge of miR-548p. The oncogenic role of circ_0008450 was partially attributed to its suppression on miR-548p. This study implies a new target for the treatment of HCC.