Exosome-derived differentiation antagonizing non-protein coding RNA with risk of hepatitis C virus-related hepatocellular carcinoma recurrence.

BACKGROUND & AIMS: Differentiation antagonizing non-protein coding RNA is associated with various types of neoplasms. Hepatitis C virus-related hepatocellular carcinoma has a high risk of recurrence. Here we determined the role of differentiation antagonizing non-protein coding RNA in hepatitis C virus-related hepatocarcinogenesis and identified potential therapeutic targets and non-invasive prognostic markers for long-term outcome of hepatitis C virus-related hepatocellular carcinoma after surgical resection. METHODS: Differentiation antagonizing non-protein coding RNAs relevant to hepatitis C virus-related hepatocellular carcinoma were identified through comparative RNA-sequencing of tumour and adjacent non-tumour (ANT) tissues in a screening set, and were validated using real-time polymerase chain reaction. Target long non-coding RNAs (lncRNAs) in tissues and serum exosomes were used to predict the recurrence of hepatitis C virus-related hepatocellular carcinoma after curative surgical resection in a large application cohort from 2005 to 2012. RESULTS: We confirmed that differentiation antagonizing non-protein coding RNA was upregulated following hepatitis C virus infection and identified as the lncRNA most relevant to hepatitis C virus-related hepatocellular carcinoma in tumour tissues as compared to that in ANT tissues. In 183 hepatitis C virus-related hepatocellular carcinoma patients followed for 10 years after curative HCC resection, the expression level of circulating exosomal differentiation antagonizing non-protein coding RNA was positively associated with HCC recurrence and was the most predictive factor associated with HCC recurrence and mortality (hazard ratio/95% confidence intervals: 7.0/4.3-11.6 and 2.7/1.5-5.1 respectively). CONCLUSIONS: Differentiation antagonizing non-protein coding RNA is highly relevant to disease progression of hepatitis C virus-related hepatocellular carcinoma. Our finding indicated that circulating exosomal differentiation antagonizing non-protein coding RNA might serve as a non-invasive prognostic biomarker for hepatitis C virus-related hepatocellular carcinoma.

The Paradoxical Effects of Different Hepatitis C Viral Loads on Host DNA Damage and Repair Abilities.

Hepatitis C virus (HCV)-induced hepatic stress is associated with increased oxidative DNA damage and has been implicated in hepatic inflammation. However, HCV infection and replication are uneven and vary among individual hepatocytes. To investigate the effect of the viral load on host DNA damage, we used an Enhanced Yellow Fluorescent Protein gene (EYFP)-tagged HCV virus to distinguish between HCV intracellular high viral load (HVL) cells and low viral load (LVL) cells. The cell sorting efficiency was confirmed by the high expression of the HCV polyprotein. We found DNA damage γ-H2AX foci in the HVL population. Comet assays demonstrated that HVL was related to the extent of the DNA strand breaks. Surprisingly, the DNA qPCR arrays and western blotting showed that the damage-related genes GPX2, MRE11, phospho-ATM, and OGG1 were significantly up-regulated in LVL cells but inversely down-regulated or consistently expressed in HVL cells. The colony survival assay to examine the repair abilities of these cells in response to irradiation showed that the LVL cells were more resistant to irradiation and had an increased ability to repair radiation-induced damage. This study found that intracellular viral loads drove cellular DNA damage levels but suppressed damage-related gene expression. However, the increase in damage-related gene expression in the LVL cells may be affected by ROS from the HVL cells. These findings provide new insights into the distinct DNA damage and repair responses resulting from different viral loads in HCV-infected cells.

GSK3beta-mediated Drp1 phosphorylation induced elongated mitochondrial morphology against oxidative stress.

Multiple phosphorylation sites of Drp1 have been characterized for their functional importance. However, the functional consequence of GSK3beta-mediated phosphorylation of Drp1 remains unclear. In this report, we pinpointed 11 Serine/Threonine sites spanning from residue 634~736 of the GED domain and robustly confirmed Drp1 Ser693 as a novel GSK3beta phosphorylation site. Our results suggest that GSK3beta-mediated phosphorylation at Ser693 does cause a dramatic decrease of GTPase activity; in contrast, GSK3beta-mediated phosphorylation at Ser693 appears not to affect Drp1 inter-/intra-molecular interactions. After identifying Ser693 as a GSK3beta phosphorylation site, we also determined that K679 is crucial for GSK3beta-binding, which strongly suggests that Drp1 is a novel substrate for GSK3beta. Thereafter, we found that overexpressed S693D, but not S693A mutant, caused an elongated mitochondrial morphology which is similar to that of K38A, S637D and K679A mutants. Interestedly, using H89 and LiCl to inhibit PKA and GSK3beta signaling, respectively, it appears that a portion of the elongated mitochondria switched to a fragmented phenotype. In investigating the biofunctionality of phosphorylation sites within the GED domain, cells overexpressing Drp1 S693D and S637D, but not S693A, showed an acquired resistance to H(2)O(2)-induced mitochondrial fragmentation and ensuing apoptosis, which affected cytochrome c, capase-3, -7, and PARP, but not LC3B, Atg-5, Beclin-1 and Bcl2 expressions. These results also showed that the S693D group is more effective in protecting both non-neuronal and neuronal cells from apoptotic death than the S637D group. Altogether, our data suggest that GSK3beta-mediated phosphorylation at Ser693 of Drp1 may be associated with mitochondrial elongation via down-regulating apoptosis, but not autophagy upon H(2)O(2) insult.