EZH2-mediated epigenetic silencing of ZIC4 in hepatocellular carcinoma

Background: The present study aimed to explore the role of ZIC4 in human liver cancer. Methods: Illumina450 genome-wide methylation data was downloaded from The Cancer Genome Atlas for 50 available liver tumor/surrounding pairs. Wound healing test, colony formation and ow cytometry assay were utilized to analyze cell migration, survival and apoptosis. The effects of EZH2 and ZIC4 on tumor growth were also investigated through in vivo xenograft and orthotopic implantation experiments. Results: ZIC4 was hypermethylated in liver cancer tissues and cell lines. EZH2 knockdown and DZNep mediated H3K27me3 contributes to ZIC4 expression. The antitumor effect of EZH2 knockdown on hepatocellular carcinoma growth, metastasis and epithelial-mesenchymal transition progression in vitro were rescued by sh-ZIC4. Downregulation of ZIC4 also rescued the antitumor effect of DZNep in vivo. Conclusions: Epigenetic silencing of ZIC4 by EZH2 mediated H3K27me3 is an important mechanism in human liver cancer and provide a new therapeutic target for the treatment of hepatocellular carcinoma disease. expression of


Introduction
Liver cancer is the fth most critical malignant tumor and the second common leading cause of cancerrelated death in males, according to statistics, there are about 782,500 people were diagnosed with liver cancer and 745,500 deaths in 2012 all over the world [1]. Hepatocellular carcinoma (HCC) has poor prognosis and is one of the most common type of liver cancer [2]. Many studies have reported that the etiological causes of HCC vary from birth and area, such as intake of alcohol in western countries, hepatitis B virus infection in Africa and East Asia, the infection of hepatitis C virus in Japan and the exposure of a atoxin B1 in Africa and China [3]. However, the decided molecular biological mechanisms that cause HCC development are still unclear, recent data suggest that the incidence of HCC is a complex process, including the activation of proto oncogenes and the inactivation of tumor inhibitor genes caused by abnormal epigenetic and genetic alterations [4]. Nevertheless, none of this information has been applied to clinical. Consequently, a deeply understanding of HCC oncogenesis and development is important to recognize corresponding molecular therapeutic mechanisms.
Epigenetic mechanisms such as histone modi cations, DNA methylation, non-coding RNAs and chromatin remodeling were proved to regulate gene expression by many cross talk ways [5]. DNA methylation is one of the most widely studied epigenetic mechanisms, which is a covalent modi cation of DNA, plays an important role in the regulation of genome function, often occurs in hepatocellular carcinoma, bladder cancer, prostate cancer and many other common cancers [6]. It was con rmed that aberrant DNA methylation of promoter CpG islands was involved with the tumor inhibitor genes silenced in many types of human cancer related diseases [7]. For example, Sun et al. illustrated that tissue suppressor of metalloproteinases3 (TIMP-3), cyclooxygenase2 (COX2), ras association domain family 1 isoform A (RASSF1A) and p16 genes are usually hypermethylated in HCC, but the methylation not occurred in non-tumor liver tissues [8]. Therefore, we proposed that DNA methylation might involve with HCC progression.
As we all known that DNA methylation is suppressed by DNA methyltransferases inhibitor such as 5-Aza-2'-deoxycytidine (5-aza-dC) [9]. Nowadays, the studies on the anti-tumor molecular mechanisms ground on epigenetic drugs are developing, 5-aza-dC has been allowed in the clinical treatment of haematological malignancies and its in uence on solid tumors were worth investigation [10]. Anwar et al. found that 5-aza-dC treatment could decrease the methylation degree and restored the expression of miR-183 [11]. EZH2, which is a histone methyltransferase, also plays a crucial role in many different kinds of cancers via epigenetic silencing of tumor inhibitor genes [12]. Previous studies showed that 3deanzaneplanocin A (DZNep), which is an inhibitor of S-adenosylhomocysteine hydrolase, could deplete PRC2 components cellular level, such as SUZ12, EED and EZH2, and further suppress the H3K27 trimethylation [13]. Therefore, we hypothesized EZH2 might involve HCC progression via H3K27me3 pathway.
It is well known that ZIC gene family function as a vital role in the development of neural crest and subsequent cerebellar [14]. Previous studies showed that ZIC gene family involved with cell proliferation regulation via suppressing neural differentiation in the dorsal neural tube, however the exact biochemical and cellular mechanism are still unknown [15]. Kandimalla et al. reported that the methylation of GATA binding protein 2 (GATA2), T-box 2 (TBX2), T-box 3 (TBX3), and Zic family member 4 (ZIC4) genes were related to the development of muscle invasive disease of papillary non-invasive bladder tumors [16]. Our study aimed to investigate the effect of ZIC4 hypermethylation on HCC progression both in vitro and in vivo.
In the present study, we investigated DNA methylation expression pro les of HCC tumor and adjacent tissues. The interactions among EZH2, H3K27me3 and ZIC4 promoter was studied, and the biological function of EZH2 and ZIC4 on HCC progression also has been explored both in vitro and in vivo. The results also pointed that epigenetic silencing of ZIC4 by EZH2 mediated H3K27me3 is an important mechanism in human liver cancer, which are very important for subsequent clinical research.

Clinical Samples
This study was approved by the institutional review board of the First A liated Hospital of Guizhou University of Traditional Chinese Medicine ethics committee and informed consent was obtained from all patients included in this study. A total of 20 Hepatocellular carcinoma patients with pairs of the tumor and the adjacent normal tissues were recruited from The First A liated Hospital of Guizhou University of Traditional Chinese Medicine, which were histologically or cytologically con rmed by at least two local pathologists. Tissues were frozen in liquid nitrogen after the surgery and stored at -80 °C.

Bioinformatic Analysis
The genome-wide methylation data of an independent dataset (The Cancer Genome Atlas) consisting of 50 HCC samples and their matched surrounding tissues were employed. The Chip Analysis Methylation Pipeline (ChAMP) package is a pipeline which not only integrates currently available 450 k analysis methods but also offers its own novel func-tionality. Statistical analyze of DNA genome methylation pro le was performed on Illumina BeadStudio software (Genetech Biotech, Taipei, Taiwan). Β values were calculated during this procedure, obtained results were selected from 0 to 0.1 to represent CpG loci, and 0-100% on behalf of the percentage of methyl-ation respectively.

Cell Culture And Drug Treatment
The human HCC-derived Hep3B, Huh-7 and HepG2 were obtained from the BeNa Culture Collection

Transfection Of Sirna
The siRNA against EZH2 and ZIC4 were synthesized by GenePharma (Shanghai, China). Transient transfections were performed by Lipofectamine 3000 cell transfection agents (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. The sh-RNA sequences are listed in Supplementary Table 1. DNA methylation analysis.
DNA was isolated using the proteinase K/phenol extraction method. Bisul te conversion was carried out using 1 µg of DNA using an Epitect Bisul te Kit (Qiagen). Bisul te-treated DNA was ampli ed with BSP primers located in the ZIC4 promoter and PCR products were cloned using the pGEM-T Easy Vector system (Promega, Madison, WI). Three individual clones were sequenced. The region assessed by BSP included 8 CpG sites from the ZIC4 promoter and average methylation from individual clones was calculated as a percentage of the number of methylated CpG sites over the number of total CpG sites sequenced.
RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR) Total RNA from frozen tissue specimens and cultured cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA quantity and quality were determined by a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington DE). The cDNA was synthesized from total RNA using PrimeScript™ RT Reagent Kit with miRNAs speci c RT primers (Takara, Dalian, China). Real-time PCR was performed on the Applied Biosystems 7300 Real-Time PCR system using SYBR Green dye (Applied Biosystems, Foster City, CA) as described by the manufacture. The relative expression levels were evaluated by using the by method. Primers for each gene were listed in Supplementary Table 1.

Chromatin Immunoprecipitation Assay
ChIP assay was performed using the EZ-CHIP chromatin immunoprecipitation kit (Millipore, Billerica, MA). followingthe manufacturer's protocol. Immunoprecipitate (IP) complexes were immunoprecipitated with an anti-EZH2 (ab191250), anti-H3K27me3 (ab192985) antibodies or rabbit IgG antibody overnight at 4 ℃. The captured genomic DNA was obtained and used for quantitative PCR analysis. Ten per cent of total genomic DNA from the nuclear extract was used as input. Ampli cation e ciency was calculated, and the data were expressed as enrichment related to input.

Wound Healing And Transwell Assays
For wound healing analysis, 24 h after the transfection, the cells were plated in 6-well plates. 24 h later, the adherent cells were wounded by a 10 µl plastic pipette tip. Then rinsing the scathing cells with PBS and culturing with serum-free DMEM for 24 h or 48 h. The wound closure in different groups was photographed and evaluated with the microscope. For the transwell assays, 1 × 10 5 cells in medium containing 0.1% FBS were seeded into the upper chamber with or without a matrigel (1:30 dilution) coated membrane (BD Biosciences, Franklin Lakes, NJ), while medium containing 10% FBS was in the under chamber. After incubation at 37 °C, gel and cells in the upper chamber were removed carefully and cells adhering to the underside of the membrane were stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) and 20% methanol. The numbers of cells were counted under an inverted microscope (Nikon).

Colony Formation Assay
24 h after the transfection, HepG2 cells were re-suspended and seeded onto 12-well plates at a density of 2,000 cells/well, incubated for two weeks, and then stained with 0.5% crystal violet for 30 min. Excess dye was rinsed off twice with phosphate-buffered saline (PBS). Images were obtained using the computer software Quantity One® from Bio-Rad Laboratories, Inc.

Flow Cytometric Assays For Apoptosis
After digested and washed twice with ice-cold PBS, cells were collected and resuspended with bindingbuffer. Cell staining was performed using AnncxinV-FITC Apoptosis Kit (Beyotime, Shanghai, China). Finally, the FACSCalibur ow analyzer (BD, CA, USA) was used to complete the detection within 30 min.

Hcc Mouse Model
Four-to ve-week-old female BALB/c nude mice were obtained from the Animal Center of the Chinese Academy of Medical Sciences (Beijing, China). Mice age 5-6 weeks were injected subcutaneously in the ank with 1◊10 7 HepG2 cells with or without ZIC4 stably knockdown. 1 week after bearing, DZNep (1 mg/Kg) was administered intraperitoneally twice per week and every 2 weeks. Tumor size was monitored by digital caliper. Tumor volume=(L◊W 2 )/2, where L is length at the widest point of the tumor and W is the maximum width perpendicular to L. Solid tumors were harvested, xed with phosphatebuffered neutral formalin, sectioned serially and stained with hematoxylin and eosin (H&E) and EZH2, ZIC4. Ki67, E-cadherin and vimentin IHC staining for standard histological examination. The subcutaneous tumor tissues were removed and implanted into the liver of nude mouse to conduct the orthotopic implantation. DZNep (1 mg/Kg) was also administered intraperitoneally twice per week and every 2 weeks. After 6 weeks, the metastases were visualized using the IVIS@ Statistical analysis SPSS 19.0 statistical software (Chicago, IL, USA) was used for statistical analysis. Quantitative data were expressed as mean ± standard deviation. A comparison of groups was analyzed by single factor ANOVA and qualitative data were expressed as the number of cases or percentage (%). A comparison of the groups was made using the χ2 test. Statistical signifcance was set at P < 0.05.

ZIC4 was hypermethylated in HCC and correlated with survival
The genome-wide methylation data of an independent dataset (The Cancer Genome Atlas) consisting of 50 HCC samples and their matched surrounding tissues were employed and found that ZIC4 was hypermethylated in tumor tissues compared to normal tissues in the top 30 candidate genes (Fig. 1A). Higher ZIC4 methylation in tumor tissues compared to paired normal tissues was displayed (Fig. 1B). Kaplan Meier analysis showed higher methylation of ZIC4 group involved with shorter survival rate (Fig. 1C). Altogether, these results suggested that ZIC4 was hypermethylated in HCC tissues and involved with poor survival rate.

Loss of ZIC4 occurs in HCC with epigenetic abnormalities
We next investigated the potential molecular mechanism that mediates the downregulation of ZIC4 in HCC. There was a typical CpG island in The ZIC4 promoter, suggesting a possible involvement of DNA methylation in the regulation of ZIC4. Although 20.8% of CpGs were methylated in LO2 cells, 62.5-91.7% CpGs were methylated in HepG2, Hep3b and Huh7 cells (Fig. 2A). The protein and mRNA levels of ZIC4 in HepG2, Hep3b and Huh7 cell lines were signi cantly downregulated (Fig. 2B). The ZIC4 expression was signi cantly increased when hepatoma cells with hypermethylated ZIC4 promoter (HepG2, Hep3b and Huh7) were treated with 5-aza-Dc for 3 days (Fig. 2C). Furthermore, ZIC4 methylation was assessed by bisul te-sequencing PCR (BSP) in 20 pairs of HCC and paracancerous tissues and ZIC4 was found to be hypermethylated in tumors tissues (Fig. 2D). ZIC4 expression was found to be downregulated in tumors tissues compared with paracancerous tissues (Fig. 2E).

EZH2-mediated H3K27me3 was involved in the repression of ZIC4 in HCC cell lines
Changes in DNA methylation could also be associated with the acquisition of histone modi cations, especially those of EZH2-dependent H3K27me3, may contribute to ZIC4 repression. As expected, ZIC4 mRNA expression were obviously upregulated in HepG2 cells treated with the sh-RNA of EZH2 and the speci c inhibitor of EZH2, DZNep (Fig. 3A-C). Furthermore, sh-EZH2 and DZNep treatment led to a decrease in the levels of H3K27me3 in HepG2 cells (Fig. 3D-E). What's more, ChIP assays showed that EZH2 occupied in the upstream region of ZIC4, which is concomitant with the increase in H3K27me3 levels ( Fig. 3F-G). These data indicate a link between epigenetic regulation and ZIC4 transcription in hepatoma cell lines.
ZIC4 mediate the effects of EZH2 on tumor promotion in vitro.
To further explore the effects of EZH2 and ZIC4, HepG2 cells were co-transfected with or without sh-EZH2 and sh-ZIC4. The protein levels of EZH2 and ZIC4 in HepG2 with or without sh-EZH2 and sh-ZIC4 treatment suggesting that EZH2 knockdown promoted ZIC4 expression and ZIC4 knockdown did not affect EZH2 expression (Fig. 4A). We performed CCK-8, anchorage-dependent colony formation and ow cytometry assays in HepG2 cells. Sh-EZH2 inhibited cell growth and promoted apoptosis, while sh-ZIC4 promoted the ability of growth and inhibited apoptosis. In addition, ZIC4 knockdown prevented the antitumor effect induced by sh-EZH2 (Fig. 4B-D). We next investigated the effect of EZH2 and ZIC4 on metastasis of HepG2 cells in vitro. Wound healing and transwell assays suggested that sh-EZH2 inhibited cell migration and invasion, while sh-ZIC4 promoted cell migration and invasion. ZIC4 knockdown rescued the antitumor effect induced by sh-EZH2 (Fig. 5A-C). What's more, ZIC4 knockdown also rescued the inhibition of EMT progression induced by sh-EZH2, as the protein levels change of E-cadherin, Ncadherin and vimentin (Fig. 5D).

ZIC4 mediate the effects of DZNep on tumor promotion in vivo.
We next investigated the effect of EZH2 and ZIC4 on tumorigenesis and metastasis in vivo. Quanti cation of tumor size and weight showed that HepG2 cells treated sh-ZIC4 generated larger tumors and HepG2 cells treated DZNep generated smaller tumors. Downregulation of ZIC4 rescued the inhibition on HepG2 cells growth induced by DZNep in vivo (Fig. 6A-C). HE staining showed more necrotic area after DZNep treatment in sh-NC or sh-ZIC4 HepG2 xenograft (Fig. 6D) and Ki67 staining also indicated lesser Ki67 positive cell after DZNep treatment in sh-NC or sh-ZIC4 HepG2 xenograft (Fig. 6E). DZNep treatment inhibited EZH2, N-cadherin and vimentin expression and promoted ZIC4 and E-cadherin expression in solid tumor (Fig. 6F). ZIC4 knockdown rescued the repressive effect of DZNep on EMT progression. In addition, for HCC orthotopic implantation mouse models, in vivo imaging system (IVIS) showed that tumor growth was signi cantly suppressed after DZNep treatment and tumor growth was signi cantly promoted after ZIC4 knockdown (Fig. 7A). H&E staining of lung tissues also showed fewer and smaller metastatic nodules in DZNep group and showed more and larger metastatic nodules in sh-ZIC4 group (Fig. 7B). ZIC4 knockdown rescued the antitumor effect induced by DZNep in vivo. The above data show that inhibitory effects of DZNep were partially mediated by sh-ZIC4 treatment.

Conclusions
In this study, we explored the role of EZH2 and ZIC4 on the physiology and regulation in HCC and found that epigenetic silencing of ZIC4 by EZH2 mediated H3K27me3 was an important mechanism in human liver cancer and it would provide a new therapeutic target for the treatment of hepatocellular carcinoma disease.

Discussion
In the present study, we found that ZIC4 was hypermethylated and down regulated in hepatocellular carcinoma cancer tissues and cells. EZH2 knockdown and DZNep resulted in low expressed of H3K27me3 while increased ZIC4 expression. The effects of EZH2 were partially mediated by ZIC4 treatment on HCC growth and metastasis in vitro and in vivo. Our study indicated that epigenetic silencing of ZIC4 by EZH2 mediated H3K27me3 is an important mechanism in human liver cancer.
Many studies have con rmed the effect of CpG island hypermethylation on the regulation of cancerassociated genes in tumorigenesis and progression [17]. For example, the methylation degree of miR-608 was higher in two cancer cells lines compared with normal cell line with the methylation level of 91.4% and 87.3% respectively, and also proposed that CpG islands hypermethylation might cause the silencing of mRNA expression [18]. In addition, Zhou et al. implicated that DBCCR1 down regulation acted as an underlying module via DNA methylation in the lung cancer pathogenesis [19]. However, it was reported that the neural transcription factors ZIC1 and ZIC4 were up regulated in desmoid tumors and other broproliferative disorder diseases, the promoter performed unmethylated in tumor tissues compared to normal tissues [20]. In the present study, we found ZIC4 was hypermethylated in HCC patients and cells, and the DNA methylation was responsible for the lower expressed mRNA, which was consistent with previous studies.
Recently, in order to decrease the methylation status of tumor inhibitor genes, epigenetic drugs are widely used to treat solid tumors with the advances of epigenetics knowledge [21]. Both DZNep treatment and EZH2 knockdown impeded anchorage-independent sphere formation and cell growth of HCC cells in culture, also pointed that and the tumor-initiating HCC cells are highly dependent on EZH2 for the tumorigenic activity [13]. Similarly, in our study, we found that EZH2 knockdown inhibited cell proliferation, migration and promoted cell apoptosis and EZH2-mediated H3K27me3 was involved in the repression of ZIC4 in HCC cell lines.
As we all known that there are ve members in ZIC gene family and each is responsible for encoding the zinc nger transcription factors, recently the mutant analysis in mice has proved the protection effect of these genes [22]. For example, ZIC1 could act as a tumor inhibitor gene and suppressed cell proliferation via inactivating p-Erk1/2 and p-Akt pathway [23]. In addition, it is also revealed the tumor inhibition effect of ZIC1 in malignant pleural mesothelioma cells [24]. Furthermore, a recent study displayed that ZIC4 was involved with the development of tumors and patients with ZIC4 hypermethylation status usually have shorter survival rate [16]. Consistent with the previous reports, we revealed that ZIC4 knockdown could promote cell proliferation, migration and inhibit cell apoptosis in vitro and promote the growth and metastasis in vivo.
In conclusion, the results of our study showed that ZIC4 is hypermethylated and downregulated in HCC tissues and cell lines. EZH2-mediated H3K27me3 was involved in the repression of ZIC4 in HCC cell lines for the rst time, which provided a new therapeutic target for the treatment of hepatocellular carcinoma disease. Availability of data and materials: All data generated or analyzed during this study are included in this article.

Supplementary Files
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