ETV4 facilitates angiogenesis in hepatocellular carcinoma by upregulating MMP14 expression.

Abnormal vascularization plays a crucial role in cell proliferation, tumor invasion and metastasis of hepatocellular carcinoma (HCC). It has been reported that ETV4 functions as an oncogenic gene in driving the carcinogenesis and progression, and promoting invasion and metastasis of HCC. However, the function of ETV4 on angiogenesis in HCC remains unclear. In the current study, immunohistochemistry showed that knockdown of ETV4 reduced angiogenesis in HCC xenograft tumor tissues. In vitro, tube formation assay verified that ETV4 expression promoted angiogenesis through simulating the angiogenic environment in HCC cells. Transcriptome sequencing indicated that MMP14 was one of the differentially expressed genes enriched in angiogenesis process. Subsequently, it was confirmed that MMP14 was regulated by ETV4 at the transcription level in HCC cells, clinical tissue samples and online databases. Further, we demonstrated that MMP14 induced angiogenesis in ETV4-mediated HCC microenvironment. Collectively, this research further reveals the biological mechanism of ETV4 in promoting the migration and invasion of HCC, and provides novel mechanistic insights and strategic guidance for anti-angiogenic therapy in HCC.

TRERNA1 upregulation mediated by HBx promotes sorafenib resistance and cell proliferation in HCC via targeting NRAS by sponging miR-22-3p.

Hepatocellular carcinoma (HCC) is among the most common malignancies and has an unfavorable prognosis. The hepatitis B virus-encoded X (HBx) protein is closely associated with hepatocarcinogenesis. Sorafenib is a unique targeted oral kinase inhibitor for advanced HCC. Long noncoding RNAs (lncRNAs) mediate HCC progression and therapeutic resistance by acting as competing endogenous RNAs (ceRNAs). However, the ceRNA regulatory mechanisms underlying sorafenib resistance in HBx-associated HCC remain largely unknown. In this study, we found that translation regulatory lncRNA 1 (TRERNA1) upregulation by HBx not only promoted HCC cell proliferation by regulating the cell cycle in vitro and in vivo but also correlated positively with poor prognosis in HCC. Importantly, TRERNA1 enhanced sorafenib resistance in HCC cells. RNA sequencing (RNA-seq) analysis indicated that NRAS proto-oncogene (NRAS) is a potential target of TRERNA1 that mediates aspects of hepatocellular carcinogenesis. TRERNA1 acts as a ceRNA to regulate NRAS expression by sponging microRNA (miR)-22-3p. In summary, we show that increased TRERNA1 expression induced by HBx reduces HCC cell sensitivity to sorafenib by activating the RAS/Raf/MEK/ERK signaling pathway. We reveal a novel regulatory mode by which the TRERNA1/miR-22-3p/NRAS axis mediates HCC progression and indicates that TRERNA1 might constitute a powerful tumor biomarker and therapeutic target in HCC.

Overexpression of eEF1A1 regulates G1-phase progression to promote HCC proliferation through the STAT1-cyclin D1 pathway.

Hepatocellular carcinoma (HCC) is a common cancer worldwide with an aggressive and highly proliferative activity. Studies had confirmed that HCC cell proliferation is associated with the cell cycle's G1 phase, but the detailed molecular mechanism has not been thoroughly elucidated to date. Eukaryotic translation elongation factor 1A1 (eEF1A1) is an evolutionarily conserved elongation factor protein and is involved in tumor cell proliferation. However, which phase of the cell cycle is regulated by eEF1A1 to influence cell proliferation in HCC and its detailed molecular mechanism remain unclear. In this study, we observed that eEF1A1 influences HCC cell proliferation by regulating the cell cycle's G1 phase. In addition, eEF1A1 influences G1 phase by regulating cyclin D1 expression, promoting HCC cell proliferation both in vitro and in vivo. Moreover, our results indicated that eEF1A1 regulates cyclin D1 expression through STAT1 signaling. STAT1 increases the transcriptional activity of cyclin D1 by binding to the cyclin D1 promoter. Taken together, these findings enabled us to identify a novel mechanism by which eEF1A1 regulates the cell cycle's G1 phase to promote tumor proliferation by regulating cyclin D1 expression through STAT1 signaling in HCC.

Rock2 promotes RCC proliferation by decreasing SCARA5 expression through ß-catenin/TCF4 signaling.

Rho-associated coiled-coil forming protein kinase 2 (Rock2), as a key effector of the small GTPase RhoA, is involved in tumor development. Scavenger receptor class A member 5 (SCARA5) is an important regulator of biological processes in cancer cells. However, the roles and relationship of Rock2 and SCARA5 in renal cell carcinoma (RCC) remain unclear. In this study, we found that Rock2 expression was markedly increased in clinical RCC tissues compared with that in adjacent non-cancerous tissues. High expression of Rock2 was inversely correlated with patient survival in RCC, which indicated that Rock2 may be a prognostic marker in human RCC. In addition, Rock2 knockdown increased SCARA5 expression and suppressed RCC cell proliferation both in vitro and in vivo. Furthermore, we found that the ß-catenin/TCF4 pathway contributed to the effect of Rock2 on SCARA5-mediated RCC proliferation. Taken together, these results suggest that this newly identified Rock2-ß-catenin/TCF4-SCARA5 axis will provide novel insight into the understanding of the regulatory mechanisms of proliferation in human RCC.

miRNA­mRNA network contributes to HBV­related hepatocellular carcinoma via immune infiltration induced by GRB2.

Chronic hepatitis B virus (HBV) infection is a critical causative factor in the tumorigenesis and progression of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) serve a critical role in the process of viral infection. However, there has been insufficient evaluation of HBV-associated miRNA-mRNA regulatory networks in HCC. The differential expression levels of miRNAs were compared in HBV-associated HCC tumor and normal tissues using the Gene Expression Omnibus database. The present study evaluated potential target genes of differentially expressed miRNAs using protein-protein interaction network, hub gene, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, gene set enrichment and immune infiltration analysis. A total of five miRNAs and seven target genes were identified in the HBV-associated miRNA-mRNA network. miRNA-93 could positively regulate the growth factor receptor bound protein 2 (GRB2) gene, while there was a positive correlation between GRB2 and cancer immune infiltrate function in Tumor Immune Estimation Resource. Collectively, the present study investigated the miRNA-mRNA regulatory network in HCC with HBV infection and showed that miRNA-93 positively regulated immune infiltration-related GRB2. Restoring GRB2 may be a candidate strategy for the treatment of HBV-related HCC.

Co-overexpression of BRD4 and CDK7 promotes cell proliferation and predicts poor prognosis in HCC.

Aberrant expression of critical components of the trans-acting super-enhancers (SE) complex contributes to the continuous and robust transcription of oncogenes in human cancers. Small-molecule inhibitors targeting core-transcriptional components such as transcriptional bromodomain protein 4 (BRD4) and cyclin-dependent kinase 7 (CDK7) have been developed and are currently undergoing preclinical and clinical testing in several malignant cancers. By analysis of TCGA data and clinical specimens, we demonstrated that BRD4 and CDK7 were frequently overexpressed in human HCCs and were associated with the poor prognosis. Shorter survival and poorly differentiated histology were linked to high BRD4 or CDK7 expression levels. Interestingly, co-overexpression of BRD4 and CDK7 was a more unfavorable prognostic factor in HCC. Treatment with JQ1 or THZ1 alone exhibited an inhibitory impact on the proliferation of HCC cells, while JQ1 synergized with THZ1 showed a more pronounced suppression. Concurrently, a combined JQ1 and THZ1 treatment abolished the transcription of oncogenes ETV4, MYC, NFE2L2. Our study suggested that BRD4 and CDK7 coupled can be a valuable biomarker in HCC diagnosis and the combination of JQ1 and THZ1 can be a promising therapeutic treatment against HCC.

HBx increases chromatin accessibility and ETV4 expression to regulate dishevelled-2 and promote HCC progression.

Hepatitis B virus (HBV) infection is the predominant causes of hepatocellular carcinoma (HCC). HBV X protein (HBx), as the most frequently integrated viral gene sequence following HBV infection, plays a critical role in the pathogenesis of HCC. H3K27ac is a characteristic marker for identifying active enhancers and even indicates chromatin accessibility associated with super-enhancers (SEs). In this study, H3K27ac ChIP-seq was applied for high-quality SE annotation of HBx-induced SEs and chromatin accessibility evaluation. The results indicated that HBx preferentially affects enrichment of H3K27ac in transcription factor signaling pathway genes, including ETV4. RNA-seq indicated that ETV4 is upregulated by HBx and that upregulated ETV4 promotes HCC progression. Interestingly, ETV4 was also included in the 568 cancer driver gene pool obtained by the Integrative OncoGenomics pipeline. However, the biological function and mechanism of ETV4 remain incompletely understood. In vivo and in vitro, we found that increased ETV4 expression promotes HCC cell migration and invasion by upregulating DVL2 and activating Wnt/ß-catenin. The mRNA and protein levels of ETV4 are higher in tumor tissues compared with adjacent tissues, and high expression of ETV4 is associated with poor prognosis in HCC patients. In summary, we first confirm that ETV4 is significantly upregulated by HBx and involved in SE-associated chromatin accessibility. Increased expression of ETV4 promotes HCC cell invasion and metastasis by upregulating DVL2. The present study provides insight into the ETV4-DVL2-ß-catenin axis in HBV-related HCC, which will be helpful for treating patients with aggressive HCC.

Targeting complexes of super-enhancers is a promising strategy for cancer therapy.

The hyperactivation and overexpression of critical oncogenes is a common occurrence in multiple types of malignant tumors. Recently, the abnormal activation mechanism of an oncogene by a super-enhancer (SE) has attracted significant attention. A series of changes (insertion, deletion, translocation and rearrangement) in the genome occurring in cancer cells may generate new SEs, leading to the overexpression of SE-driven oncogenes. SEs are composed of typical enhancers densely loaded with mediator complexes, transcription factors, and chromatin regulators, and drive the overexpression of oncogenes associated with cellular identity and disease. Cyclin-dependent kinase 7 (CDK7) and bromodomain protein 4 (BRD4) are critical mediator complexes associated with SE-mediated transcription. Clinical trials have shown that emerging small-molecule inhibitors (CDK7 and BRD4 inhibitor), targeting the SE exert a notable effect on cancer treatment. Increasing evidences has illustrated that the SE and its associated complexes play a critical role in the development of various types of cancer. The present review discusses the composition, function and regulation of SEs and their contribution to oncogenic transcription. In addition, creative therapeutic approaches that target SE, their advantages and disadvantages, as well as the problems with their clinical application are discussed. It was found that targeting SE may be used in conventional treatment and establish more access for patients with cancer.

High methylation levels of histone H3 lysine 9 associated with activation of hypoxia-inducible factor 1α (HIF-1α) predict patients' worse prognosis in human hepatocellular carcinomas.

Although it is becoming increasingly apparent that histone methyltransferases and histone demethylases play crucial roles in the cellular response to hypoxia, the impact of hypoxic environments on global patterns of histone methylation is not well demonstrated. In this study, we try to detect the global levels of histone lysine methylation in HCC cases and analyze the correlation between these modifications and the activation of hypoxia-inducible factor 1α (HIF-1α). Immunohistochemistry was used to detect the global levels of histone H3 lysine 9 dimethylation (H3K9me2), histone H3 lysine 9 trimethylation (H3K9me3), histone H3 lysine 27 trimethylation (H3K27me3) and the nuclear expression of HIF-1α in tissue arrays from 111 paraffin-embedded HCC samples. Our analyses revealed that the global levels of H3K9me2, H3K9me3 and the nuclear expression of HIF-1α were distinctly higher in HCC tissues than in peritumoral tissues. Both H3K9me2 and H3K9me3 were positively correlated with the degree of tumor differentiation and the patients' prognosis. Analysis based on the Pearson's correlation coefficient indicated a positive correlation between H3K9me2 and the nuclear expression of HIF-1α, and meanwhile, a significant correlation between the expression of H3K9me2 and H3K9me3 was also found. In addition, the combination of H3K9me2, H3K9me3 and HIF-1α, rather than one single histone modification or molecular maker, is a better prognostic maker for HCC patients. These findings provide new insights on the complex networks underlying cellular and genomic regulation in response to hypoxia and may provide novel targets for future therapies.

[Eukaryotic translation elongation factor 1A1 positively regulates NOB1 expression to promote invasion and metastasis of hepatocellular carcinoma cells in vitro].

OBJECTIVE: To explore the role of eukaryotic translation elongation factor 1A1 (eEF1A1) in regulating the invasion and metastasis of hepatocellular carcinoma (HCC) cells and the possible mechanism. METHODS: qRT-PCR and Western blotting were used to detect the mRNA and protein expression of eEF1A1 and NOB1 in different HCC cell lines and normal liver cells. The invasion and migration abilities of HCC cells with eEF1A1 knockdown or overexpression were examined using Transwell chamber assay and RTCA assay, and the changes in NOB1 mRNA and protein expressions in the cells were detected. The effects of increasing NOB1 expression in HCCLM3-sheEF1A1 cells and decreasing NOB1 expression in eEF1A1-overexpressing MHCC97h cells on eEF1A1 expression and cell invasion and migration abilities were analyzed using Western blotting, Transwell chamber assay and RTCA assay. RESULTS: The expressions of eEF1A1 and NOB1 were significantly increased in positive correlation in HCC cells as compared with normal hepatocytes. Knockdown of eEF1A1 significantly decreased the invasion and migration of HCC cells and reduced the mRNA and protein expression of NOB1 (P < 0.01). Overexpression of eEF1A1 significantly enhanced invasion and migration of HCC cells and increased NOB1 mRNA and protein expressions (P < 0.01). Increasing NOB1 expression in HCCLM3-sheEF1A1 cells led to the restoration of NOB1 expression and cell invasion and migration abilities (P < 0.01), whereas decreasing NOB1 in MHCC97h-eEF1A1 cells resulted in inhibition of NOB1 expression and cell invasion and migration (P < 0.01). CONCLUSIONS: eEF1A1 positively regulates the expression of NOB1 to promote the invasion and migration of HCC cells in vitro.

lncRNAs as prognostic molecular biomarkers in hepatocellular carcinoma: a systematic review and meta-analysis.

The latest studies have shown that long non-coding RNAs (lncRNAs) may be considered markers as their expression levels were abnormal in cancer and can be used as a molecular biomarker for the potential assessment of cancer prognosis. In this study, we aimed to assess the prognostic value of lncRNA as marker of patients with hepatocellular carcinoma. We performed a detailed search of the PubMed and Embase databases for articles on the prognostic value of various lncRNAs in HCC. We then carefully extracted the relevant data from the articles, and we used the meta-analysis method to analyze these results; heterogeneity and publication bias were also evaluated. With 40 associative studies included, we found that high expression of 27 types of lncRNA was associated with a poor prognosis in HCC patients, and low expression of 18 types of lncRNAs was associated with a worse prognosis. Patients with higher lncRNA expression had significantly poor overall survival (OS; pooled HR, 1.25; 95% confidence interval [CI], 1.03-1.52) as well as significantly poor recurrence-free survival (RFS; pooled HR, 1.66; 95% CI, 1.26-2.17). Overexpression of lncRNAs may not meaningfully predict disease-free survival (DFS; pooled HR, 1.04; 95% CI, 0.52-2.07; p = 0.91). Our meta-analysis demonstrated that lncRNAs may serve as predictive biomarkers for cancer prognosis.