Deoxyribonuclease 1-like 3 Inhibits Hepatocellular Carcinoma Progression by Inducing Apoptosis and Reprogramming Glucose Metabolism

HCC has remained one of the challenging cancers to treat, owing to the paucity of drugs targeting the critical survival pathways. Considering the cancer cells are deficient in DNase activity, the increase of an autonomous apoptisis endonuclease should be a reasonable choice for cancer treatment. In this study, we investigated whether DNASE1L3, an endonuclease implicated in apoptosis, could inhibit the progress of HCC. We found DNASE1L3 was down-regulated in HCC tissues, whereas its high expression was positively associated with the favorable prognosis of patients with HCC. Besides, serum DNASE1L3 levels were lower in HCC patients than in healthy individuals. Functionally, we found that DNASE1L3 inhibited the proliferation of tumor cells by inducing G0/G1 cell cycle arrest and cell apoptosis in vitro. Additionally, DNASE1L3 overexpression suppressed tumor growth in vivo. Furthermore, we found that DNASE1L3 overexpression weakened glycolysis in HCC cells and tissues via inactivating the rate-limiting enzymes involved in PTPN2-HK2 and CEBPβ-p53-PFK1 pathways. Finally, we identified the HBx to inhibit DNASE1L3 expression by up-regulating the expression of ZNF384. Collectively, our findings demonstrated that DNASE1L3 could inhibit the HCC progression through inducing cell apoptosis and weakening glycolysis. We believe DNASE1L3 could be considered as a promising prognostic biomarker and therapeutic target for HCC.

Those cell lines were cultured in high-glucose DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA). All cells were cultured in an incubator at 37 °C with 5% CO 2 . All patients were routinely examined every 3-6 months during the first five years of follow-up and once a year thereafter. The overall survival time was defined as the period in months from operation to death. The disease-free survival time was defined as the period in months from operation to the date at which relapse was clinically identified. These patients' data were used to analyze the correction between glucose metabolism and serum DNASE1L3 activity.

Total RNA isolation and quantitative real-time PCR (qRT-PCR)
TRIzol Reagent was used to separate total RNA from tumor tissues and cells. Reverse transcription of mRNA was conducted with PrimeScript RT Master Mix (Takara, Japan). Quantitative real-time PCR was performed using SYBR qPCR Mix (Toyobo, Osaka, Japan) and a CFX Connect Real-Time PCR Detection System (Bio-Rad, USA). The relative gene expression was measured using the 2-ΔΔCT method. The GAPDH was used as an internal control.  Supplementary Table 1.

Immunohistochemistry (IHC) and immunofluorescence (IF)
The tumor tissues were collected and fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to 4 µm thickness. Then, the tissue sections were exposed to the antigen by incubation with dewaxed water and at 100 °C for 15 min. The tissues were incubated with 3% H 2 O 2 for 10 min at 37 °C to block endogenous peroxidase activity, following incubation with BSA and primary antibody overnight at 4 °C. After incubation with HRP-labeled secondary antibodies, HRP was detected with standard substrates for subsequent detection. The sections were stained with hematoxylin and dehydrated with graded alcohol and xylene.
For immunofluorescence, the cells were washed twice and fixed with 4% paraformaldehyde, then permeabilized with 0.5% Triton X-100, then the cells were incubated with primary and secondary antibodies according to the manufacturer's protocol. The coverslips were counterstained with DAPI and imaged with Olympus FV1000 (Tokyo, Japan).

Cell proliferation assay
After cells were transfected for 48 h with the desired siRNA or plasmid, cell proliferation was measured using CCK8 assay according to the manufacturer's instruction. For colony formation assay, the cells transfected after 48 h were seeded (1000/well) in 6-well plates for 7-10 days. Colonies (≥50 cells/colony) were calculated after fixation with 4% paraformaldehyde and stained with crystal violet solution.

Cell migration and invasion assay
Cell migration and invasion were measured using the scratch assay. After transfection with desired plasmid or siRNA, about 10 6 cells were seeded into 6-well plates under serum-free conditions and the scratches were

Western blotting
Protein samples were prepared using RIPA cell lysis buffer with protease inhibitors and phosphatase inhibitors (Roche, Mannheim, Germany). The total protein quantity was measured using the BCA assay. The samples were separated via 10% SDS-PAGE and were transferred onto PVDF membranes, and incubated at 4 °C overnight with primary antibodies against the indicated proteins on a rotating wheel. After incubation with HRP-conjugated secondary antibodies for 1 h, antibody-binding proteins were detected using Clarity TM Western ECL Substrate (Bio-Rad, Hercules, CA, USA). The information on antibodies used in our study is provided in Supplementary Table 2.

Chromatin immunoprecipitation assay
In order to detect the interaction between the DNA and proteins, chromatin immunoprecipitation (ChIP) assay was performed using the Magna ChIP-seq™ Chromatin Immunoprecipitation Kit (Millipore; Billerica, USA) according to manufacturer's instructions. ZNF384 or IgG antibody and protein A/G beads were incubated overnight at 4 °C.
Afterward, DNA was extracted from the DNA-protein complex. The immunoprecipitation DNA was verified by ChIP-PCR.

Tumorigenesis assay
Five-week-old male BALB/c nude mice were purchased from the Animal Center of the Chinese Academy of Medical Sciences (Beijing, China).
Guidelines for Care and Use of Laboratory Animals were followed during the investigation. All animal procedures were approved by the Ethics Committee of the Zhongnan Hospital of Wuhan University. All animals were maintained under specific pathogen-free conditions. For tumor growth analysis, HCCLM3 cells (1×10 6 /100 µL PBS) that stably overexpressed DNASEL13 or control were subcutaneously injected into the armpits of six nude mice per group. The tumor volume was calculated every five days. The mice were sacrificed six weeks after injection, and the tumors were dissected and imaged. To verify the intrahepatic antitumor effect, an orthotopic HCC mouse model was established. First, the subcutaneous tumors of mice were implanted based on the protocols mentioned above. Then tumors were collected when they reach 1 cm in diameter and cut into pieces of about 1 mm 3 in size under aseptic conditions. After washing with PBS, the tumors were waterlogged in the serum-free medium. Afterward, the nude mice were anesthetized, and then a midline abdominal incision was made to expose the liver, subsequent one piece of tumor was implanted into the left liver lobe of each mouse. Finally, a suture compression was done to stop bleeding, and the abdominal incision was sutured. After six weeks, the mice were sacrificed to dissect liver tumors for the following experiments.

Label-free quantitative proteomics
Label-free quantitative proteomics was carried out by GeneCreate Biological Engineering Co., Ltd. (Wuhan, China). The mass spectrometry data were collected using Q Exactive (Thermo Fischer Scientific, Waltham, MA, USA) and a liquid-mass system. Protein lysate was added to the cell precipitate and then incubated on ice for 5 min. DTT with a final concentration of 10 mM was added, followed by an ice bath with ultrasound for 15 min, then 13,000 g was centrifuged at 4 °C for 20 min. After the protein solution was diluted five times with 100 mM TEAB, trypsin was added at a mass ratio of 1:50 (trypsin: protein) and digested overnight at 37 °C. The peptides after enzymatic hydrolysis were desalted with the C18 column, and the peptides after vacuum freeze-drying were desalted.

Metabolite quantification by liquid chromatography-tandem mass spectrometry (LC-MS/MS)
Metabolite quantification was conducted by Biotree Biotech (Shanghai, China). Briefly, 80% HPLC-grade methanol (cooled to −80 °C) was added to the frozen cells and vortexed for 1 min at 4 °C and incubated for 4 h at −80 °C, and then centrifuged at 14,000 g for 15 min using a refrigerated centrifuge with 4 °C. The supernatant was transferred to a new 1.5 mL tube, and speedy Vac/lyophilize for LC-MS analysis. For quantification of absolute metabolites concentrations, U-1 3 C-glutamine was used as an internal standard and was added to the extraction buffer containing 80% methanol. All samples and standards were measured using a TSQ VANTAGE interfaced with Ultimate 3000 Liquid Chromatography system (Thermo Fischer Scientific), equipped with a HILIC HPLC carbon column (Amide 4.6 × 100 mm ID 3.5 µm; Part No:186004868, Waters). Mobile phase buffer A (1L) contained 20 mM ammonium hydroxide, 20 mM ammonium acetate, 95% water and 5% (v/v) acetonitrile, pH 9.0 at 25 °C. While the mobile phase buffer B contained 100% acetonitrile. The column was maintained at a controlled temperature of 30 °C and was equilibrated with 15% buffer A for 3 min at a constant flow rate of 350 µL/min. 10 µL aliquots of each sample were loaded onto the column, and the compounds were eluted from the column with a linear gradient of 15-50% buffer A from the 4th to 16th min, and then increased to 85% buffer A from 16th min to 20th min and decreased to 15% buffer A from 21th to 23th min, and the column was washed for 2 min with 15% Buffer A. The ion transfer tube temperature was set to 350 °C and vaporizer temperature was 270 °C. The instrument was run in negative mode with a spray voltage of 3000 sheath gas 40 and Aux gas 5.0. About seven to eight concentrations (from low to high) of the different standard mixtures were measured using multiple reactions monitoring mode (MRM) with optimal collision energies to produce a standard curve.

Hematoxylin-eosin (H&E) staining
The collected tumor samples from the mice liver were fixed in 4% paraformaldehyde. The tissues were dehydrated, embedded in paraffin, and cut into slices. The sections were stained with hematoxylin and eosin and observed by light microscopy (Olympus, Tokyo, Japan).

DNASE1L3 Elisa Assay
Natural coagulation of blood was performed by centrifuging the blood sample at 2000-3000 rpm for 10-20 min at room temperature. The purified DNASE1L3 antibody was used to coat the microporous plate.
Standard materials and serum samples to be tested were added to the microporous plate in turn and then combined with the HRP-labeled DNASE1L3 antibody to form the antibody-antigen-enzyme labeled antibody complex. After thorough washing, the substrate TMB was added for color development. TMB turns blue after reacting with the HRP enzyme and then into yellow after the addition of hydrochloric acid. The color intensity was positively correlated with DNASE1L3 in the sample.
The absorbance value (O.D. value) was measured with an enzyme marker at 450 nm, and the concentration of DNASE1L3 in the sample was calculated using a standard curve.