Background: The gut-liver axis plays a pivotal role in the pathogenesis of hepatocellular carcinoma (HCC). However, the correlations between the gut microbiome and the liver tumor transcriptome in patients with HCC and the impact of the gut microbiota on clinical outcome are less well-understood. Methods: Fecal samples collected from HBV-related HCC patients (n = 113) and healthy volunteers (n = 100) were subjected to 16S rRNA sequencing of the microbiome. After a rigorous selection process, 32 paired tumor and adjacent non-tumor liver tissues from the HCC group were subjected to next-generation sequencing (NGS) RNA-seq. The datasets were analyzed individually and integrated with clinical characteristics for combined analysis using bioinformatics approaches. We further verified the potential of the gut microbiota to predict clinical outcome by a random forest model and a support vector machine model. Results: We found that Bacteroides, Lachnospiracea incertae sedis, and Clostridium XIVa were enriched in HCC patients with a high tumor burden. By integrating the microbiome and transcriptome, we identified 31 robust associations between the above three genera and well-characterized genes, indicating possible mechanistic relationships in tumor immune microenvironment. Clinical characteristics and database analysis suggested that serum bile acids may be important communication mediators between these three genera and the host transcriptome. Finally, among these three genera, six important microbial markers associated with tumor immune microenvironment or bile acid metabolism showed the potential to predict clinical outcome (AUC = 81%). Conclusions: This study revealed that changes in tumor immune microenvironment caused by the gut microbiota via serum bile acids may be important factors associated with tumor burden and adverse clinical outcome. Gut microbes can be used as biomarkers of clinical features and outcomes, and the microbe-associated transcripts of host tumors can partly explain how gut microbiota promotes HCC pathogenesis.

Long non-coding RNA HOTAIR exerts regulatory functions in various biological processes in cancer cells, such as proliferation, apoptosis, mobility, and invasion. We previously found that HOX transcript antisense RNA (HOTAIR) is a negative prognostic factor and exhibits oncogenic activity in hepatocellular carcinoma (HCC). In this study, we aimed to investigate the role and molecular mechanism of HOTAIR in promoting HCC cell migration and invasion. Firstly, we profiled its gene expression pattern by microarray analysis of HOTAIR loss in Bel-7402 HCC cell line. The results showed that 129 genes were significantly down-regulated, while 167 genes were significantly up-regulated (fold change >2, p < 0.05). Bioinformatics analysis indicated that RNA binding proteins were involved in this biological process. HOTAIR suppression using RNAi strategy with HepG2 and Bel-7402 cells increased the mRNA and protein expression levels of RNA binding motif protein 38 (RBM38). Moreover, the expression levels of RBM38 in HCC specimens were significantly lower than paired adjacent noncancerous tissues. In addition, knockdown of HOTAIR resulted in a decrease of cell migration and invasion, which could be specifically rescued by down-regulation of RBM38. Taken together, HOTAIR could promote migration and invasion of HCC cells by inhibiting RBM38, which indicated critical roles of HOTAIR and RBM38 in HCC progression.

Minichromosome Maintenance family (MCMs), as replication licensing factors, is involved in the pathogenesis of tumors. Here, we investigated the expression of MCMs and their values in hepatocellular carcinoma (HCC). MCMs were analyzed in 105 samples including normal livers (n = 15), cirrhotic livers (n = 40), HCC (n = 50) using quantitative polymerase chain reaction (qPCR) (Cohort 1). Significantly up-regulated MCMs were verified in 102 HCC and matched peritumoral livers using PCR (Cohort 2), and the correlations with clinical features and outcomes were determined. In addition, the focused MCMs were analyzed in parallel immunohistochemistry of 345 samples on spectrum of hepatocarcinogenesis (Cohort 3) and queried for the potential specific role in cell cycle. MCM2–7, MCM8 and MCM10 was significantly up-regulated in HCC in Cohort 1. In Cohort 2, overexpression of MCM2–7, MCM8 and MCM10 was verified and significantly correlated with each other. Elevated MCM2, MCM6 and MCM7 were associated with adverse tumor features and poorer outcomes. In Cohort 3, MCM6 exhibited superior HCC diagnostic performance compared with MCM2 and MCM7 (AUC: 0.896 vs. 0.675 and 0.771, P < 0.01). Additionally, MCM6 other than MCM2 and MCM7 independently predicted poorer survival in 175 HCC patients. Furthermore, knockdown of MCM6 caused a delay in S/G2-phase progression as evidenced by down-regulation of CDK2, CDK4, CyclinA, CyclinB1, CyclinD1, and CyclinE in HCC cells. We analyze MCMs mRNA and protein levels in tissue samples during hepatocarcinogenesis. MCM6 is identified as a driver of S/G2 cell cycle progression and a potential diagnostic and prognostic marker in HCC.

The deregulation of microRNAs has been reported to play a pivotal role in hepatocellular carcinoma (HCC). MiR-126-3p has been reported to be associated with poor prognosis in HCC. However the underlying mechanism of miR-126-3p in HCC remains unclear. The expression levels of miR-126-3p in HCC tissues and cells were detected by RT-PCR. Transwell assay and capillary tube formation assay were applied to assess the metastasis and angiogenesis in vitro. Nude mice subcutaneous tumor model was used to perform in vivo study. Dual- luciferase reporter assay was conducted to confirm the direct binding of miR-126-3p and target genes. The changes of biomarker protein levels were examined by western blot and Immunohistochemistry. We observed that the miR-126-3p expression levels in HCC tissues and cells were significantly down-regulated. Through gain- and loss- of function studies, we showed that miR-126-3p dramatically inhibited HCC cells from migrating and invading extracellular matrix gel and suppressed capillary tube formation of endothelial cells in vitro. Furthermore, overexpression of miR-126-3p significantly reduced the volume of tumor and microvessel density in vivo. LRP6 and PIK3R2 were identified as targets of miR-126-3p. Silencing LRP6 and PIK3R2 had similar effects of miR-126-3p restoration on metastasis and angiogenesis individually in HCC cells. Furthermore, the miR-126-3p level was inversely correlated with LRP6 and PIK3R2 in HCC tissues. In addition, the rescue experiments indicated that the metastasis and angiogenesis functions of miR-126-3p were mediated by LRP6 and PIK3R2. Our results demonstrates that deregulation of miR-126-3p contributes to metastasis and angiogenesis in HCC. The restoration of miR-126-3p expression may be a promising strategy for HCC therapy.

MicroRNA-452 promotes stem-like cells of hepatocellular carcinoma by inhibiting sox7 involving wnt/β-catenin signaling pathway

Background/Aims: Doxorubicin-eluting bead TACE (DEB-TACE) has recently been developed as a novel therapy option for HCC. However, the clinical efficacy of DEB-TACE is still unclear. Herein, we performed a meta-analysis to evaluate the efficacy of DEB-TACE compared with conventional TACE (cTACE). Methodology: We inlcuded seven studies (a total of 693 patients) to compare DEB-TACE with cTACE. The pooled odds ratios (OR) were calculated using a random or fixed effects model. MEDLINE, EMBASE and the Cochrane Database were searched for articles published from dates of inceptions up to February 20, 2012. Sensitivity analysis and publication bias estimate were also performed to evaluate the potential risk bias in the overall results of pooled analysis. Results: The pooled estimates for tumor response of DEB-TACE were not significantly different from those of cTACE, with CR (OR: 1.18; 95%CI: 0.81-1.71; p=0.394), PR (OR: 1.37; 95%CI: 0.94-1.99; p=0.101), SD (OR: 0.88; 95%CI: 0.51-1.51; p=0.637), PD (OR: 0.85; 95%CI: 0.52-1.38; p=0.512), DC (OR: 1.37, 95%CI: 0.95-1.98; p=0.089) and OR (OR: 1.40; 95%CI: 0.97-2.000; p=0.070). Conclusions: The current evidence suggests that DEB-TACE is able to accomplish the same tumor response as cTACE. Although this analysis provides a comprehensive look at published data involving the clinical efficacy of DEB-TACE compared with conventional TACE, additional large scale of randomized-control studies are still needed.