Requirement of cyclin-dependent kinase function for hepatitis B virus cccDNA synthesis as measured by digital PCR.

INTRODUCTION AND OBJECTIVES: HBV covalently closed circular (ccc) DNA is the key player in viral persistence and an important predictive biomarker for hepatitis relapse. Precise quantification of intracellular cccDNA is challenging because cccDNA is present in very low levels in hepatocytes, where it also co-exists with a large excess amount of relaxed circular (rc) DNA. We aimed to develop a highly sensitive cccDNA detection method for cccDNA quantification by digital PCR (dPCR). PATIENTS OR MATERIALS AND METHODS: A standard plasmid containing the whole HBV genome in the closed circular conformation was employed to characterize the performance of dPCR. rcDNA in the growth medium of HBV-producing HepAD38 cells was used as a matrix for cccDNA detection. Intrahepatic cccDNA measurement by dPCR and qPCR was performed to determine the correlation of the analysis results for the two methods. RESULTS: The limit of detection (LOD) of the cccDNA dPCR was 1.05copy/µl, and the linear range of detection was 1.02×104copies/µl, achieving a dynamic detection range of 104-fold. cccDNA measurement using excess rcDNA as the matrix did not reveal false-positive detection, indicating that dPCR was highly specific. In the HepAD38 cells, the cccDNA levels measured by dPCR were highly correlated with those measured by qPCR but had a higher sensitivity. The CDK inhibitor AZD-5438 was found to block intracellular cccDNA synthesis. CONCLUSIONS: Dpcr greatly improved the sensitivity and specificity of cccDNA detection. Host CDK activities are likely required for cccDNA synthesis. dPCR can potentially be applied for drug screening for effective cccDNA inhibitors.

NEK2 Promotes Hepatoma Metastasis and Serves as Biomarker for High Recurrence Risk after Hepatic Resection.

INTRODUCTION AND AIM: Developing reliable biomarkers for hepatocellular carcinoma (HCC) patients who are at a high risk of recurrence after curative hepatic resection is very important for determining subsequent therapeutic strategies. We investigated the role of the cell cycle factor NIMA-related kinase 2 (NEK2) in HCC progression in hepatoma cells and post-surgery patients. MATERIAL AND METHODS: The effects of NEK2 on proliferation, invasion and migration of hepatoma HuH7 and SK-Hep1 cells were evaluated. In a post-surgery HCC cohort (N = 97), the Nek2 induction levels in the tumors were examined with real-time RT-PCR analysis, and the results were analyzed for their correlations with recurrence. RESULTS: NEK2 promoted G1 to S phase cell cycle progression by causing increases in cyclin D1 and AKT phosphorylation and decreases in the cyclin-dependent kinase inhibitor p27, indicating that NEK2 plays an important role during interphase in addition to its previously identified role in M phase. NEK2 also enhanced the proliferation, migration and invasion of hepatoma cells and regulated the expression of E-cadherin and MMP9. The Nek2 mRNA levels in the tumors were highly correlated with recurrence rates in the post-surgery HCC patients. Combined evaluation of the tumor AJCC stage and the Nek2 level can serve as a reliable method for predicting the relative risk of HCC recurrence in these patients. CONCLUSIONS: NEK2 plays a significant role in cell cycle progression in the inter- and M-phases. NEK2 enhances HCC metastasis and is correlated with recurrence and thus can potentially serve a promising high-risk biomarker for HCC.