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.     

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.     

A novel multidimensional signature predicts prognosis in hepatocellular carcinoma patients

The abnormal expression of microRNAs (miRNAs) or protein-coding genes (PCGs) have been found to be associated with the prognosis of hepatocellular carcinoma (HCC) patients. Using bioinformatics analysis methods including Cox’s proportional hazards regression analysis, the random survival forest algorithm, Kaplan–Meier, and receiver operating characteristic (ROC) curve analysis, we mined the gene expression profiles of 469 HCC patients from The Cancer Genome Atlas (n = 379) and Gene Expression Omnibus (GSE14520; n = 90) public database. We selected a signature comprising one protein-coding gene (PCG; DNA polymerase μ) and three miRNAs (hsa-miR-149-5p, hsa-miR-424-5p, hsa-miR-579-5p) with highest accurate prediction (area under the ROC curve [AUC] = 0.72; n = 189) from the training data set. The signature stratified patients into high- and low-risk groups with significantly different survival (median 27.9 vs. 55.2 months, log-rank test, p < 0.001) in the training data set, and its risk stratification ability were validated in the test data set (median 47.4 vs. 84.4 months, log-rank test, p = 0.03) and an independent data set (median 31.0 vs. 46.0 months, log-rank test, p = 0.01). Multivariable Cox regression analysis showed that the signature was an independent prognostic factor. And the signature was proved to have a better survival prediction power than tumor–node–metastasis (TNM) stage (AUC _signature = 0.72/0.64/0.62 vs. AUC _TNM = 0.65/0.61/0.61; p < 0.05). Moreover, we validated the expression of these prognostic genes from the PCG-miRNA signature in Huh-7 cell by real-time polymerase chain reaction. In conclusion, we found a signature that can predict survival of HCC patients and serve as a prognostic marker for HCC.     

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.     

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.     

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.     

MicroRNA-33b regulates hepatocellular carcinoma cell proliferation,apoptosis, and mobility via targeting Fli-1-mediated Notch1 pathway

MicroRNAs (miRNAs) have been confirmed to play pivotal roles in hepatocellular carcinoma (HCC) carcinogenesis. However, the underlying function of microRNA-33b (miR-33b) in HCC remains unclear. Here, we found that miR-33b level was significantly reduced in both HCC tissues and tumor cell lines. Further, luciferase reporter assay and western blot analysis confirmed that Friend leukemia virus integration 1 (Fli-1) was a direct target of miR-33b. Overexpression of miR-33b dramatically suppressed HCC tumor cell proliferation and cell mobility, but facilitated tumor cell apoptosis in vitro. Besides, restoration of Fli-1 partially attenuated miR-33b-mediated inhibition of cell growth and metastasis via activating Notch1 signaling and its downstream effectors. Our findings demonstrate the important role of miR-33b/Fli-1 axis in HCC progression and provide novel therapeutic candidates for HCC clinical treatment.     

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.     

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.     

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.     

Knockdown of PSMC3IP suppresses the proliferation and xenografted tumorigenesis of hepatocellular carcinoma cell

Hepatocellular carcinoma (HCC) is the fifth most frequent malignancy and the second leading cause of cancer-related death worldwide. Proteasome 26S subunit ATPase 3 interacting protein (PSMC3IP) is an oncogene in breast cancer, while its role in HCC remains unclear. Here, we found that PSMC3IP was critical for the cell proliferation and tumorigenic capacity of HCC cells. Upregulation of PSMC3IP was observed in HCC specimens, and high PSMC3IP expression predicted poor overall survival of HCC patients. In vitro, knockdown of PSMC3IP blunted the proliferation and colony formation of BEL-7404 and SMMC-7721 cells. Likewise, PSMC3IP silencing suppressed the xenografted tumor development of BEL-7404 cells. Mechanistically, apoptosis was enhanced after PSMC3IP knockdown in both BEL-7404 and SMMC-7721 cells. At the molecular level, TP53 and GNG4 were upregulated and eukaryotic translation initiation factor 4E (EIF4E) and insulin like growth factor 1 receptor (IGF1R) were downregulated in shPSMC3IP compared with shCtrl BEL-7404 cells. Therefore, targeting PSMC3IP maybe a promising strategy for HCC.     

四逆散对人肝癌HepG2细胞增殖、凋亡的影响及其机制

目的:探讨含四逆散药液血清对人肝癌HepG2细胞增殖、凋亡的影响及机制。方法:将人肝癌HepG2细胞分为5组,每组3个复孔。实验组细胞用五氟尿嘧啶(5-FU)或不同浓度的含四逆散药液血清处理48 h后,用倒置显微镜观察含四逆散药液血清处理后人肝癌HepG2细胞形态的变化; MTT法检测含四逆散药液血清对HepG2细胞生长的抑制作用;荧光染色和流式细胞术分别分析含四逆散药液血清对HepG2细胞凋亡的影响。Rho123染色法检测线粒体膜电位变化,Western blot检测细胞凋亡相关蛋白的表达。结果:与对照组比较,含四逆散药液血清处理人肝癌HepG2细胞后,细胞数量显著减少(P<0.01),形态发生改变,呈现典型的凋亡细胞形态; G1期细胞数明显增加,而G2期细胞数量显著减少(P<0.05); Bax、Caspase-3、-9和Cyt-c的表达显著升高,而Bcl-2的表达显著降低(P<0.05);随着含四逆散药液血清浓度增大,HepG2细胞线粒体膜电位显著下降(P<0.05)。结论:四逆散可以抑制HepG2细胞增殖,并通过线粒体途径诱导细胞凋亡。