RETRACTED ARTICLE: Dickkopf-1 contributes to hepatocellular carcinoma tumorigenesis by activating the Wnt/β-catenin signaling pathway

Dysregulation of dickkopf-related protein 1 (DKK1) expression has been reported in a variety of human cancers. We previously reported that DKK1 was upregulated in hepatocellular carcinoma (HCC). However, the role of DKK1 in HCC remains unclear. This study aimed to investigate the clinical significance and biological functions of DKK1 in HCC. The expression of DKK1 was examined in cirrhotic and HCC tissues by immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). DKK1 was silenced or overexpressed in HCC cell lines, and in vitro and in vivo studies were performed. Immunohistochemistry revealed that DKK1 was weakly expressed in cirrhotic tissues (8/22, 36.4%) but upregulated in HCC tissues (48/53, 90.6%, cohort 1). Significant upregulation of DKK1 was observed in 57.6% (19/33, cohort 2) of HCC tissues by qRT-PCR, and the expression of DKK1 was associated with tumor size (P = 0.024) and tumor number (P = 0.019). Genetic depletion of DKK1 impaired the proliferation, colony-forming ability, invasion, and tumor formation of HCC cells (HepG2 and HUH-7). Conversely, forced expression of DKK1 increased the proliferation, colony-forming ability, and invasion of HepG2 and HUH-7 cells in vitro and enhanced tumor formation in vivo. Subsequent investigation revealed that the DKK1-mediated proliferation and tumorigenicity of HepG2 and HUH-7 cells is dependent on the Wnt/β-catenin signaling pathway. These findings indicate that DKK1 plays an oncogenic role in HCC by activating the Wnt/β-catenin signaling pathway.


1.Supplementary Information
The protein levels of DKK1 in DKK1-shRNA and LV-DKK1 HCC cells were quantified by ImageJ (a, b). The protein levels of Wnt1 and β-catenin in DKK1-shRNA and LV-DKK1 HCC cells were quantified by ImageJ(c, d). The protein levels of nuclearβ-catenin in DKK1-shRNA and LV-DKK1 HCC cells were quantified by ImageJ (e). The protein levels of β-catenin in β-catenin-shRNA HCC cells were quantified by ImageJ (f). *P<0.05

2.Supplementary Materials and Methods
Ethics approval and consent to participate Informed consent was obtained from all subjects according to the Internal Review and Ethics Boards of Sun Yat-Sen Memorial Hospital, and the project was in accordance with the Helsinki Declaration of 1964. All animal experimentation described in this study was performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at Sun Yat-sen University.

Patients and immunohistochemistry
Samples of cirrhotic and tumor tissues were used in tissue microarray assays (TMAs).
These samples were obtained from twenty-two cirrhotic patients and fifty-three consecutive HCC patients who underwent resection at the Department of Hepato-Pancreato-Biliary Surgery, Sun Yat-sen Memorial Hospital. Thirty-three pairs of fresh tumor tissues and the corresponding peritumoral tissues were randomly chosen from the tissue bank of Sun Yat-sen Memorial Hospital and were used for qRT-PCR analysis. None of the patients received chemotherapy or radiation therapy prior to the radical tumor resection. The differences and significance of DKK1 expression in these patients were investigated. The clinical and histopathological data, including gender, age, functional state of the liver, size of the tumor and tumor number, are presented in Table 1 Quantitative RT-PCR and Western Blot analysis Total RNA was isolated using RNAiso Plus reagent according to the manufacturer's protocol (TaKaRa, Tokyo, Japan). Primer sets used for PCR amplification were shown in supplement Table 2. As a control, the levels of glyceraldehyde phosphate dehydrogenase (GAPDH) expression were also analyzed. Real-time PCR was performed as described. 25 Total protein extraction and western blot analysis was also performed as described in our previous study. 25 Briefly, Protein lysates obtained from the cultured cells were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were probed with primary antibodies recognizing DKK1 (1:2000), WNT1 (1:2000), β-catenin (1:5000) and β-Tubulin (1: 1000). After incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson Immunoresearch, USA), protein bands were visualized using enhanced chemiluminescence (ECL) plus Western blotting detection reagents followed by exposure to Hyper-films (Amersham, UK). Details regarding the primary and secondary antibodies are provided in Supplementary Table 1.

Nuclear protein extraction was performed as described in the Supplementary
Materials and Methods. The results of western blot were quantified by Image J (Supplementary Fig. 3).

Nuclear protein extraction
Nuclear protein extraction was performed as follows:

Cell proliferation assay and colony Formation Assay
Cells (5 × 10 3 /well) were cultured in 96-well tissue culture plates until they reached 50% confluence. Cell viability was determined using a Cell Counting Kit-8 (CCK-8) purchased from Dojindo Molecular Technologies (Gaithersburg, MA). Briefly, 10 μl of water-soluble formazan dye was added to each well and incubated for 2 h. The absorbance at 450 nm was measured by an enzyme linked immunosorbent assay (ELISA) plate reader. The absorbance of the negative control (OD) was considered to be 0.
HCC cells were plated in triplicate in six-well plates. After 7 days, the cells were rinsed with PBS twice, fixed with 10% formaldehyde, and stained with 0.1% crystal violet in 10% ethanol and the numbers of colonies were counted.

Cell cycle analysis
The cell cycle was analyzed using flow cytometry with propidium iodide (PI; Sigma, USA) staining. For each group, the cells were harvested and washed with phosphate-buffered saline (PBS) and fixed overnight in ice-cold 70% ethanol. The cells were washed twice with PBS and treated with 1 mg/L RNaseA (TaKaRa, Japan) for 15 min. Finally, the cells were stained with 50 mg/L PI in the dark for 1 h. Then, the cell cycle analysis was performed using a fluorescence-activated cell sorter (BD Biosciences, Franklin Lakes, NJ, USA), and the PI fluorescence was measured at 488 nm. Each group was analyzed in triplicate, and at least 10,000 cells were analyzed in each experiment.

Cell invasion assay
The invasive activity of HepG2 and SMMC-7721 cells was estimated using transwell plates (6.5 mm in diameter, polycarbonate membrane, 8 μm pore size) coated with extracellular matrix gel obtained from Corning (Corning, NY, USA). Twenty-four hours after transfection, an aliquot of 1 × 10 5 cells was placed in the upper chamber with 0.1 ml of serum-free medium, whereas the lower chamber (of a 24-well plate) was loaded with 0.5 ml of medium containing 10% fetal bovine serum. After 24 h of incubation, the cells were fixed with 4% paraformaldehyde and then counterstained with 0.1% crystal violet. The cells that had migrated into the lower chambers were observed and counted under a light microscope. Then, the number of migratory cells was calculated.

In vivo subcutaneous xenografts assay
Briefly, 5 × 10 6 cells were suspended in 100 µl PBS and were injected subcutaneously into 4-weeks old female nude mice (Balb/c nu/nu). Tumor volumes were monitored every 7 days by measuring the length and width with a caliper and using the formula (width 2 ) × length/2. Mice were sacrificed 8 weeks after injection, and the tumors were isolated and measured. All animals in our laboratory received care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals, prepared by the National Academy of Sciences and published by the National Institutes of Healthy.