Downregulation of UHRF1 increases tumor malignancy by activating the CXCR4/AKT-JNK/IL-6/Snail signaling axis in hepatocellular carcinoma cells

UHRF1 (ubiquitin-like, with PHD and RING finger domains 1) plays a crucial role in DNA methylation, chromatin remodeling and gene expression and is aberrantly upregulated in various types of human cancers. However, the precise role of UHRF1 in cancer remains controversial. In this study, we observed that hypoxia-induced downregulation of UHRF1 contributes to the induction of the epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma cells. By negatively modulating UHRF1 expression, we further showed that UHRF1 deficiency in itself is sufficient to increase the migratory and invasive properties of cells via inducing EMT, increasing the tumorigenic capacity of cells and leading to the expansion of cancer stem-like cells. Epigenetic changes caused by UHRF1 deficiency triggered the upregulation of CXCR4, thereby activating AKT and JNK to increase the expression and secretion of IL-6. In addition, IL-6 readily activated the JAK/STAT3/Snail signaling axis, which subsequently contributed to UHRF1 deficiency-induced EMT. Our results collectively demonstrate that UHRF1 deficiency may play a pivotal role in the malignant alteration of cancer cells.


Tumor spheroid formation and immunocytochemistry
To generate tumor spheroids under non-adherent conditions, the cells were seeded in 24-well ultra-low-attachment plates (Corning Costar Corp., Cambridge, MA, USA) at 3000 cells per well and cultured for 5 days. Immediately after harvesting, the spheroids were fixed and then embedded in OCT compound (Scigen Scientific Inc., Gardena, CA, USA). Afterward, the spheroids that were embedded in OCT compound were sectioned using a Cryotome FSE cryostat (Thermo Scientific) into 10 μm thick in accordance with the manufacturer's instructions, and stained overnight at 4°C with anti-UHRF1 (1:500) and -HIF1α (1:500) antibodies. After washing with PBS, 1:200 dilution of TEXAS RED-conjugated goat antirabbit IgG or anti-mouse IgG antibody in blocking solution was applied to the cryosections, and the samples were incubated for 60 min. The nuclei were counterstained with DAPI. The stained paraffin sections were observed and imaged using a confocal microscope (Carl Zeiss, Thornwood, NY, USA).

In vivo tumor growth
All animal protocols used in this study were approved by the Institutional Animal Care and Use Committee at Dongnam Institute of Radiological & Medical Sciences (DIRAMS; Busan, Republic of Korea). Female BALB/c nude (6-weeks old) were purchased from Japan SLC, Inc.(Shizuoka, Japan). Total number of 16 nude mice were randomly divided into two groups (shCont and shUHRF1; n=8 for each group) and each cell line (1 × 10 7 ) was inoculated subcutaneously into nude mice. Tumor volume was estimated as follows: tumor volume = (short axis) 2 × (long axis) × 0.5. All animal studies were followed by a blind randomized animal study protocol.

Tail vein injection
The cells (1 × 10 6 ) were injected into tail veins of athymic BALB/C nude mice and allowed tumor formation for 2 months. When tumor was formed in the lung, the lung tissues were sectioned for immunohistochemistry. This study was reviewed and approved by the Institutional Animal Care and Use Committee at Dongnam Institute of Radiological & Medical Sciences (DIRAMS; Busan, Republic of Korea).

Immunohistochemistry
Athymic BALB/C nude mice were killed and tumor tissues were harvested and fixed in 4% formaldehyde, followed by cryoprotection with 30% sucrose for 72 h at 4°C. Afterward, the tissues were cut using a Cryotome FSE cryostat (Thermo Scientific) into 10 μm thick sections in accordance with the manufacturer's instructions. Sections were stained with H&E (hematoxylin and eosin). Additionally, the cells were imaged by phase contrast microscopy (Nikon Eclipse 80i; Nikon, Tokyo, Japan).

DNA methylation analysis via bisulfite sequencing (BSP)
Genomic DNA was extracted from cells using TRIZOL (Gibco), and then subjected to sodium bisulfite conversion using the EZ DNA Methylation-Gold Kit (Zymo Research Corp., Orange, CA, USA) according to the manufacturer's instructions. The bisulfite-converted genomic DNA was used for the methylation analysis of the CpG island of CXCR4 promoter (-1326 bp ~ -986 bp) with the PCR primer for DNA methylation. The primer for BSP was designed by using the MethPrimer program (http://www.urogene.org/methprimer/). The primer was as follows: forward (5′-TATTAGGGAGGGGTTTTAGATAAAG-3′) and reverse (5′-CCAAAAATAAACAAAAATTCCAAAC-3′). Afterward, the amplified products were cloned into the pGEMT-T easy vector (Promega, Madison, WI), and then DNA sequencing was performed on 10 individual clones (Macrogen, Seoul, Korea).

Sphere forming assay
Cells were grown in serum-free DMEM/F12 (Gibco) supplemented with B27 (Gibco), N2 (Gibco), 20 ng/mL basic fibroblast-(Peprotech, London, UK) and 20 ng/mL epidermal growth factor (Peprotech) onto 24-well ultra low attachment plates at 300 cells per well for 7 or 14 days, and then the size and number of spheres were determined using a phase-contrast Nikon microscope (TS100; Tokyo, Japan). To measure the size of sphere, 12 spheres per group were randomly selected.

Fluorescence-activated cell sorting
The cells were detached from the dishes with trypsin-EDTA (Gibco), counted, and washed in PBS (phosphate-buffered saline) containing 0.1% BSA (Bovine serum albumin) (Santa Cruz Biotechnology Inc.). 1 × 10 7 cells in 500 μl PBS containing 0.5% BSA were incubated with APC-conjugated fluorescence-labeled mouse anti-human CD133 (Miltenti Biotec, Bergisch Gladbach, Germany) or APC-conjugated isotype control mouse IgG2b (BD Biosciences) at      The cells were grown in the presence or absence of SP600125 (5 μM) for 48 h, and western blot analysis was performed. β-actin was used as a loading control. Results from three independent experiments are expressed as means ± SEMs. (* P < 0.05, ** P < 0.01). Figure S5. UHRF1 deficiency-induced activation of AKT and JNK contributes to an increase in the sphereforming ability of HepG2 cells.

Supplementary figure legends
(a) Quantification of sphere-forming abilities of shCont-and shUHRF1-Hep3B cells. The cells were grown in DMEM/F12 supplemented with B27, N2, basic fibroblast-and epidermal growth factor onto 24-well ultra low attachment plates at 500 cells per well for 7 days, and the size of spheres were determined. To measure the size of sphere, 12 spheres per group were randomly selected. (b) Western blot analysis for CD133 in shCont-and shUHRF1-Hep3B cells. (c) Effect of siRNA targeting Snail1 on the sphere-forming ability of shUHRF1-HepG2 cells. The cells were grown in the presence or absence of siRNA targeting Snail1 for 48 h, and sphere-forming assay was performed for 7 days. (d and e) Effect of SP600125 or LY294002 on the sphere-forming ability of shUHRF1-HepG2 cells. The cells were grown in the presence or absence of SP600125 (5 μM) or LY294002 (2 μM) for 48 h, and sphere culture was performed for 7 days. To measure the size of sphere, 12 spheres per group were randomly selected. Results from three independent experiments are expressed as means ± SEMs. (* P < 0.05, ** P < 0.01) Figure S6. UHRF1 is downregulated in sphere-forming-or CD133 + HepG2 cells. (a) A comparison of the protein expression of UHRF1 in monolayered and sphere-forming HepG2 cells. Sphere culture of HepG2 cells was performed for 7 days, and the protein expression of UHRF1 in sphere-forming HepG2 cells was compared with that in monolayered HepG2 cells using western blot analysis. (b) The isolation of CD133and CD133 + cells from HepG2 cells. The dot plot is divided into four quadrants for CD133cells (Q1+Q3) and CD133 + cells (Q2+Q4) which were isolated by flow cytometry sorting. (c) A comparison of the mRNA expression level of UHRF1 in CD133and CD133 + cells. After isolation of CD133and CD133 + cells, qRT-PCR analysis was performed. β-actin was used as a loading control. Results from three independent experiments are expressed as means ± SEMs. (* P < 0.05, ** P < 0.01).