C-terminal truncated hepatitis B virus X protein regulates tumorigenicity, self-renewal and drug resistance via STAT3/Nanog signaling pathway

Hepatitis B virus (HBV) is a major risk factor of chronic liver disease and hepatocellular carcinoma (HCC). Random integration of HBV DNA into the host genome is frequent in HCC leading to truncation of the HBV DNA, particularly at the C-terminal end of the HBV X protein (HBx). C-terminally truncated HBx (HBx-ΔC) has been implicated in playing a pro-oncogenic role in hepatocarcinogenesis. However, the mechanism whereby HBx-ΔC1 contributes to hepatocarcinogenesis remains unclear. In this study, we investigated the functional role of HBx-ΔC1 in regulating liver cancer stem cell (CSC) properties. Using Tet-on inducible system, we found that HBx-ΔC1 enhanced CSC properties including self-renewal, tumorigenicity, chemoresistance, migration and expression of liver CSC markers, when compared with the full-length HBx counterpart and vector control. Interestingly, HBx-ΔC1 conferred resistance in HCC cells towards sorafenib treatment through suppression of apoptotic cascade. In addition, HBx-ΔC1 upregulated a panel of stemness genes, in which Nanog was found to be among the most significant one in both trasnfected cell lines. Consistently, Nanog was upregulated in human HCC samples which had HBx-ΔC1 expression. Furthermore, the induction of CSC properties by HBx-ΔC1 was via the Stat3/Nanog pathway, as administration of Stat3 inhibitor abolished the HBx-ΔC1-induced self-renewing capacity. In conclusion, our data suggest that HBx-ΔC1 enhances liver CSCs properties through Stat3/Nanog cascade, and provide a new insight for the therapeutic intervention for HBV-related HCC.


Quantitative PCR (qPCR) analysis
Total RNA was isolated using Trizol reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). Complementary DNA (cDNA) was synthesized using a GeneAmp® Gold RNA PCR Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions and then subjected to PCR with a SYBR Green PCR kit with primers specific to the sequences of which are provided in Supplementary Methods. The amplification protocol consisted of incubations at 94°C for 15 seconds, 63°C for 30 seconds, and 72°C for 60 seconds. Incorporation of the SYBR Green dye into PCR products was monitored in real time with an ABI 7900HT Sequence Detection System and SDS 1.9.1 software (Applied Biosystems) and subsequently analyzed using RQ Manager 1.2 software (Applied Biosystems), thereby allowing the threshold cycle (C T ) at which exponential amplification of the products began to be determined. The amount of target cDNA was calculated relative to that of β-actin cDNA.

Migration and invasion assays
Cell migration was investigated using transwell inserts with 6.5 mm polucarboate membranes of 8.0μm pore size (Corning Inc., NY, USA). Cells in serumfree medium were seeded onto the upper chamber of the transwell and the culture medium containing 10% FBS was added into the lower chamber serving as chemoattractant. Cells were incubated for 24hr to allow for migration through the membrane. Migrated cells were then fixed by 2% paraformaldehyde and stained with crystal violet. Stained cells were captured randomly for three fields under a light microscope and ells were counted. The experiments were repeated independently three times. The cell invasion assay was performed with self-coated Matrigel (BD Biosciences, San Jose, CA) on the upper surface of a transwell chamber. The invasive cells that had invaded through the extracellular matrix layer to the lower surface of the membrane were fixed with 2% paraformaldehyde and stained with crystal violet. Photographs of three randomly selected fields of the fixed cells were captured and cells were counted. The experiments were repeated independently three times.

In vivo tumorigenicity experiments
Tumorigenicity was evaluated in vivo by subcutaneous xenograft tumor models. Various number of HCC cells were subcutaneously injected at 4 sites (2 in the right flank and 2 in the left flank) into the nonobese diabetic (NOD)/ immune-deficient (SCID) mice to induce xenograft tumor formation. After injection, mice were administrated with 0.2mg/ml doxycycline (BD Bioscience) dissolved in drinking water containing 2% sucrose, and were under observation for tumor growth up 8 wk.

Flow cytometry analysis
Phycoerythrin (PE)-conjugated CD133, CD47 and CD24 cell surface markers or its isotype-matched mouse immunoglobulins (BD Biosciences, San Jose, CA; Miltenyl Biotec, Auburn, CA) were used to stain cells in freshly prepared phosphate-buffered saline (PBS) containing 2% FBS at 4°C for 30-45 min. Samples were then washed with PBS and analyzed with FACSCantoII flow cytometer and FACSDiva software (BD Biosciences, San Jose, CA) with a minimum acquisition of 10,000 events per sample.

Annexin V/propidium iodide apoptosis assay
Apoptosis, or programmed cell death, was determined by using Propidium iodide (PI) in conjunction with Annexin V. Cells were stained with FITC-conjugated Annexin V and PI at 5μl and 10μl per test, respectively, in 1x Annexin V binding buffer from FITC Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose, CA) in dark at room temperature for 15 min. Apoptotic cells were detected and analyzed using FACSCantoII flow cytometer and FACSDiva software (BD Biosciences, San Jose, CA).