Hepatocellular carcinoma (HCC) is a common malignancy that has region specific etiologies. Unfortunately, 85% of cases of HCC are diagnosed at an advanced stage. Reliable biomarkers for the early diagnosis of HCC are urgently required to reduced mortality and therapeutic expenditure. We established a non-targeted gas chromatography–time of flight–mass spectrometry (GC-TOFMS) metabolomics method in conjunction with Random Forests (RF) analysis based on 201 serum samples from healthy controls (NC), hepatitis B virus (HBV), liver cirrhosis (LC) and HCC patients to explore the metabolic characteristics in the progression of hepatocellular carcinogenesis. Ultimately, 15 metabolites were identified intimately associated with the process. Phenylalanine, malic acid and 5-methoxytryptamine for HBV vs. NC, palmitic acid for LC vs. HBV, and asparagine and β-glutamate for HCC vs. LC were screened as the liver disease-specific potential biomarkers with an excellent discriminant performance. All the metabolic perturbations in these liver diseases are associated with pathways for energy metabolism, macromolecular synthesis, and maintaining the redox balance to protect tumor cells from oxidative stress.

MicroRNAs (miRNAs) are stably present in human serum. The relationship between circulating miRNAs and hepatitis B virus (HBV) infected liver disease has not been previously reported. Applied Biosystems array-based miRNA expression profiling was performed on pooled sera obtained from identified groups of chronic asymptomatic carriers (ASC), patients with chronic hepatitis B (CHB) and HBV-associated acute-on-chronic liver failure (ACLF), as well as healthy controls (HC). Nine miRNAs were verified in more clinical samples by RT-PCR. The correlation between miRNAs expression and the relationship between miRNA levels and clinical characteristics was analysed. Results showed that circulating miRNAs were detected in all disease and control samples, and their numbers increased with symptom severity, from 37 in HC, 77 in ASC, 101 in CHB, to 135 in ACLF. The expression levels of most miRNAs were also up-regulated in HBV-infected patients when compared to HC. Expression of the liver-specific miR-122 was significantly up-regulated in HBV-infected patients. Concomitant regulation of miRNAs not in clusters was disrupted by HBV infection. However, such disruption was not observed for miRNAs in paralogous clusters. Furthermore, the level of miRNAs in the CHB serum was up-regulated most in hepatitis B e antigen-positive patients. The expression levels of miR-122 and miR-194 correlated negatively with the age of patients with CHB or ACLF. Functional analysis showed that miR-122 could inhibit HBV replication in Huh7 and HepG2 cells. In all, our study revealed that a number of miRNAs were differentially expressed during HBV infection and underscored the potential importance of miR-122 in the infection process.

Hydrogen sulfide (H 2 S), in its gaseous form, plays an important role in tumor carcinogenesis. This study investigated the effects of H 2 S on the cell biological functions of hepatocellular carcinoma (HCC). HCC cell lines, HepG2 and HLE, were treated with NaHS, a donor of H 2 S, and rapamycin, a classic autophagy inducer, for different lengths of time. Western blotting, immunofluorescence, transmission electron microscopy (TEM), scratch assay, CCK-8 and flow cytometric analysis were carried out to examine the effects of H 2 S on HCC autophagy, cell behavior and PI3K/Akt/mTOR signaling. Treatment with NaHS upregulated expression of LC3-II and Atg5, two autophagy-related proteins, in HepG2 and HLE cells. TEM revealed increased numbers of intracellular doublemembrane vesicles in those cells treated with NaHS. Like rapamycin, NaHS also significantly inhibited expression of p-PI3K, p-Akt and mTOR proteins in HCC cells. Interestingly, the expression of LC3-II was further increased when the cells were treated with NaHS together with rapamycin. In addition, NaHS inhibited HCC cell migration, proliferation and cell division. These findings show that H 2 S can induce HCC cell apoptosis. The biological function of the gasotransmitter H 2 S in HCC cells was enhanced by the addition of rapamycin. Hydrogen sulfide influences multiple biological functions of HCC cells through inhibiting the PI3K/Akt/ mTOR signaling pathway.