C-terminal truncated HBx initiates hepatocarcinogenesis by downregulating TXNIP and reprogramming glucose metabolism

Chronic hepatitis B virus (HBV) infection is strongly associated with the initiation and development of hepatocellular carcinoma (HCC). However, the genetic alterations and pathogenesis mechanisms remain significantly unexplored, especially for HBV-induced metabolic reprogramming. Analysis of integration breakpoints in HBV-positive HCC samples revealed the preferential clustering pattern within the 3′-end of X gene in the HBV genome, leading to the production of C-terminal truncated X protein (Ct-HBx). In this study, we not only characterized the oncogenic role of two Ct-HBx (HBx-120 and HBx-134) via in vitro and in vivo functional assays but also deciphered their underlying molecular mechanisms. Gene expression profiling by transcriptome sequencing identified potential targets of Ct-HBx and novel malignant hallmarks such as glycolysis, cell cycle, and m-TORC1 signaling in Ct-HBx-expressing cells. TXNIP, a well-established regulator of glucose metabolism, was shown to be downregulated by Ct-HBx and play a pivotal role in Ct-HBx-mediated HCC progression. Suppression of TXNIP is frequently observed in HCC patients with Ct-HBx expression and significantly (P = 0.015) correlated to a poorer prognosis. Re-introduction of TXNIP attenuated the metabolic reprogramming induced by the Ct-HBx and inhibited the tumor growth in the mice model. Further study suggested that Ct-HBx could downregulate TXNIP via a transcriptional repressor nuclear factor of activated T cells 2 (NFACT2). Collectively, our findings indicate that TXNIP plays a critical role in Ct-HBx-mediated hepatocarcinogenesis, serving as a novel therapeutic strategy in HCC treatment.

was clearly visible. Cells were washed with PBS 3 times followed by fixed with 4% paraformaldehyde for 15 minutes, then stained with 1% crystal violet (Sigma) for 15 minutes before washed carefully under tap water. The colonies were counted using light microscopy. All results in this assay are based on triple independent repeats at least.

Migration and invasion assay
For migration assay, cells were trypsinized and resuspended in the serum-free medium, diluted the cell to a certain concentration. Added 750 uL DMEM medium supplemented with 10% FBS to the lower chamber and grew cells at the appropriate density (For LO2 cells, 3×104/well; for MIHA cells, 2×104/well) in 600 uL FBS free DMEM at the upper chamber (Falcon). Cells were cultured for 24-72 hours and collected; fixed following stained according to the same protocol as colony formation assay.
For invasion assay, wells (Falcon) were pre-treated by adding 500 uL FBS-free DMEM in the upper and lower chamber and incubated for 2 hours at 37°C. Other steps were the same as migration assay, except for the cell density (For LO2 cells, 6×104/well; for MIHA cells, 4×104/well).

Mice model
Cells were pre-treated and suspended in PBS at proper concentration(For LO2 cells, 4×107/mL; for MIHA cells, 108/mL ), 4-week-old BALBc/nude male mice (n=6 for treatment groups) under anesthetization were subcutaneously injected into both sides of flank with 100uL cells stably transfected with HBx or TXNIP expression plasmids (mice from the same batch were randomly allocated to each group), MIHA cells were mixed with Matrigel (50:100 cell suspension ) before inoculation to increase the tumor formation rate. Mice after surgery were monitored every three days and tumor size and weight of mice were recorded by a blinded investigator. 4-6 weeks later, mice were sacrificed, and part of the fresh tumors was collected for RNA extraction and others were fixed in 4% paraformaldehyde for paraffin-embedded specimens.

Western blotting
The western blotting analysis was performed strictly followed the standard protocols of antibodies of TXNIP, TPI1, LDHA, PHD (Cell signaling technology, CST), IDH1, MDH2, CS(Santa Cruz Biotechnology), HIF1a (GeneTex), mTOR, p-mTOR (Abcam) and b-actin (Santa Cruz Biotechnology) is used as the internal reference. The whole-cell protein was extracted and standardized.

Immunohistochemical (IHC) staining
Cells attached to an 8-well chamber or paraffin-embedded tissue slides were used for IHC staining. Briefly, for tissue slides, paraffin-embedded sections (6um) were deparaffinized and rehydrated by immersing the sections into xylene, 1:1 mix of xylene and pure ethanol, each for 5 mins at room temperature, followed by 95%, 75%, 50% ethanol each for 3 mins at room temperature. The slides then would be kept in tap water until put into 95°C retrieving buffer (10X Antigen Retrieval Buffer, diluted in ddH2O) for 45mins. For cell chambers, cells were prepared the same as IF protocol and washed in PBS followed by fixed with ice-cold 4% paraformaldehyde for 15 minutes. Then for both cell chamber and tissue slides, the endogenous peroxidase activity was blocked with 3% H2O2 for 1 hour at room temperature followed by washing in PBS 3 times; then normal goat serum was incubated for 2 hours to reduce nonspecific binding. Without washing, chambers or sections were incubated with primary antibody against TXNIP (1:100 dilution, Abcam) at 4°C overnight in a humidified box. Then slides were washed in PBS 3 times and incubated with biotinylated goat anti-rabbit IgG (Santa Cruz) for 30min at 37°C and streptavidin-peroxidase conjugated for 15min at room temperature. Finally, the 3, 5diaminobenzidine (DAB) Substrate Kit (Dako) was used for color development followed by Mayer's hematoxylin counterstaining.

Immunofluorescence staining assay (IF)
Cells attached on the 8-well chamber were used for IF staining. Briefly, cells were seeded on chamber at 70% confluence and allowed to attach overnight. Then the chamber slides were rinsed into PBS 3 times and then fixed with ice-cold 4% paraformaldehyde for 15 minutes. Then, cells on the chamber slides were permeabilized in 0.1% Triton X-100 (Sigma-Aldrich, prepared in PBS) for 15 minutes at room temperature, followed by blocking with 5% BSA (prepared in PBS) at room temperature for 30 minutes. The slides would then be incubated with antibodies against the flag (1:100, CST) overnight at 4C. The secondary antibodies (Abcam) were used at the concentration of 1:400 in dark for 1hour at room temperature. At the last step, the slides were rinsed into PBS 3 times and stained with DAPI at the concentration of 1/1000 for 10 mins at room temperature and mounted with mounting gel for IF staining.

RNA extraction, Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR)
For qRT-PCR analysis in cell lines and clinical samples, total RNA was extracted using RNAisoPlus (TAKARA BIO INC) and cDNA was synthesized by reverse transcription using PrimeScript™ RT reagent Kit (TAKARA). Then qRT-PCR was performed using a SYBR Green PCR Kit (TAKARA)and corresponding primers, Ct values were detected by ABI PRISM 7900 Sequence Detector, and data were processed using SDS 1.9.1 software. The sequence of primers are listed in Supplementary Table 1

Dual-Luciferase Reporter Assay
Briefly, around 2.5 kb (-2kb-500bp) of the promoter sequence of TXNIP were amplified (primer For the serial dual-luciferase reporter assay which will be applied to identify the precise binding site on TXNIP promoter, a series of truncated mutants of TXNIP promoter(sequence of primers is listed in Supplementary Table 2) will be generated and cloned into pGL3-enhancer vector separately, and a serial of TXNIP promoter activity will be analyzed.
For site-directed mutagenesis of the potential binding motif, NFATC2, STAT3, FOXO1, and MYOG binding sites in the TXNIP promoter were mutated with a QuikChange II site-directed mutagenesis kit (Stratagene) (sequence of primers is listed in Supplementary Table. S2). The mutation of these constructs was confirmed by DNA sequencing.

Plasmid construction
Different lengths of HBV X genes with flag-tag encoding for the full-length HBx (HBx-154

Chromatin immunoprecipitation (CHIP) assay
Briefly, DNA and the binding protein are cross-linked using formaldehyde, and lysate will be sonicated to shear DNA to the length between 200bp to 500 bp. Then the compound will be immunoprecipitated with the antibody against the cis-regulatory element. Protein A agarose will be used to pull down the antibody-chromatin complexes. Complexes incubated with IgG are used as the negative control. Also, input will be needed without any antibody incubation. After crosslinking reversal and proteinase K treatment, DNAs will be purified by phenol-chloroform extraction and ethanol precipitation, then amplified by qRT-PCR with specific primers (sequence of primers are listed in Supplementary Table. S1) on the predicted binding site on TXNIP promoter.

Cell cycle analysis
Briefly, cells were seeded in 6-well plate at proper density (For LO2 cells, 2×105/mL; for MIHA cells, 106/mL) and allow to attach overnight, then cells were starved by cultured in FBS-free DMEM for 6-36 hours. Cells were collected at different time points (including the dead cells in the supernatant), washed in PBS, fixed with cold 70% alcohol and stained with propidium iodide (PI). Flow cytometry would be applied to determine the cell cycle distribution.

Statistical analysis
Results were presented as mean ± SEM, Student's t-test was used to compare the mean value of two groups. Pearson χ2 test was applied to determine the correlation of TXNIP expression and the clinical parameters in HCC patients. Wilcoxon rank-sum test was used to compare the distribution pattern of HBV breakpoints between HCC samples and normal liver tissues.
Statistical significance was defined as P<0.05.
For clinicopathologic analysis, the Chi-square test were applied to analyze the correlation between truncated and full-length HBx and TXNIP in clinical samples. A comparison of the