Downregulation of histone methyltransferase SET8 inhibits progression of hepatocellular carcinoma

The expression of lysine methyltransferase SET8, which is involved in carcinogenesis of many types of human cancers through monomethylation of histone H4 lysine 20 (H4K20), is associated with the prognosis of hepatocellular carcinoma (HCC). We performed a functional analysis for SET8 to assess its effect on HCC progression. SET8 knockdown inhibited proliferation, migration and invasion of HCC cells. SET8 knockdown also inhibited tumour growth in a human xenograft mouse model. Overexpression of SET8 displayed the reverse effect, while treatment with the SET8 inhibitor UNC0379 produced an effect similar to SET8 knockdown. In addition, drug sensitivity testing in SET8-siRNA transfected HCC cells indicated that docetaxel inhibited cell growth dramatically, as demonstrated by the Cell Counting Kit-8 (CCK-8) assay. Furthermore, gene expression microarray analysis showed that genes altered after SET8 knockdown were clustered in pathways related to tumorigenesis and metastasis. Our data suggests that targeting SET8 for HCC therapy can inhibit the proliferation and invasion of HCC cells as well as increase their sensitivity to chemotherapy.

Wound healing assay. Cells were cultured in 6-well plates. A scratch was created on the cell layer using a 200 µl pipette tip when cells reached 100% confluence. Subsequently, cells were washed twice with PBS. Images of the cells in each group were captured at 0, 12, 24 and 48 h after scratching. At least five fields were analysed for each scratch and cell migration rates were calculated using the following formula 18 .  In vivo green fluorescent imaging was performed using the NightOwl Bioimager (Berthold Technologies, Bad Wildbad, Germany) once a week. Signal intensity was quantified using the WinLight32 software package (Berthold Technologies).
Statistical analysis. Data are expressed as means ± standard deviation. Statistical analysis between two groups was performed with Student's t test, and the comparison between three or more groups was performed with One-way ANOVA. P < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 19.0 software package (SPSS Inc., Chicago, IL).
To further evaluate the in vivo effect of SET8 knockdown on tumour growth, the growth rate of SMMC-7721 xenografts stably transfected SET8-siRNA was compared to xenografts transfected with control-siRNA. As shown in Fig. 1H,I, the growth of SET8-siRNA xenografts was significantly decreased compared with that of control-siRNA xenografts, as assessed with green fluorescent imaging. The tumour volume of SET8-siRNA xenografts was smaller than that of control-siRNA xenografts at 14, 21 and 28 days after implantation (P < 0.05) according to calliper measurement. These results suggest that SET8 knockdown inhibited growth of SMMC-7721 cells in vivo.

Overexpression of SET8 promotes proliferation, migration and invasion of HCC cells. The effect
of SET8 overexpression on HCC progression in terms of proliferation, migration and invasion was evaluated in Huh-7 cells. Cells transfected with SET8-pEZ-M61 promoted proliferation from 24 to 72 h (P < 0.05, Fig. 2A,B), migration (P < 0.05, Fig. 2C,D) at 24 and 48 h), and invasion (P < 0.01, Fig. 2E) compared with cells transfected with pEZ-M61 or blank control cells. SET8 overexpression not only reduced p53 expression, but also increased p53K382mel (Fig. 2F). These data demonstrated that overexpression of SET8 promoted proliferation, migration and invasion of HCC cells.

An inhibitor of SET8 inhibits proliferation, migration and invasion of HCC cells. UNC0379, a
selective and substrate-competitive inhibitor of SET8 22 , was used to evaluate whether it mimics the effect of SET8 siRNA on HCC progression. SMMC-7721 and Huh-7 cells were treated with different concentrations of UNC0379 (0, 5 or 10 µM) for 72 h. Our results showed that growth of SMMC-7721 and Huh-7 cells was dramatically inhibited by UNC0379 (Fig. 3A,B). UNC0379 inhibited proliferation from 24 to 72 h (P < 0.01, Fig. 3C,D,E,F), migration (P < 0.05, Fig. 3G,H,I,J) at 24 and 48 h, and invasion (P < 0.01, Fig. 3K,L) in these two cell lines. This inhibitor also mimicked the effect of SET8 siRNA regarding the increase in p53 levels and the decrease in p53K382mel (Fig. 3M). These data demonstrated that SET8 was an effective target for HCC prevention.  www.nature.com/scientificreports www.nature.com/scientificreports/ Gene expression profile of SET8-knockdown cells. To investigate the target genes induced by SET8 knockdown in HCC cells, we monitored the changes in mRNA levels between SET8-siRNA cells and control-siRNA cells with whole-genome expression microarray analysis (Fig. 4A). A total of 406 differentially expressed genes were identified, including 181 upregulated and 225 downregulated genes (fold change> 2.0, q < 0.05) (Fig. 4B, Supplementary Table S1). Biological network analysis for these 406 differentially expressed genes with Reactome FI showed that enrichment of biological processes related to the cell cycle, cell proliferation, cell migration, cell adhesion, apoptosis, cytokine, angiogenesis and the enriched signalling pathways, namely p53 signalling pathway 23 , Wnt signalling pathway 24 and VEGF signalling pathway 25 , were associated with SET8 knockdown (Fig. 4C, Supplementary Table S2). These data demonstrated that SET8 modified the progression of HCC by modulating these signalling pathways.

Discussion
In the present study, we evaluated the function of SET8 in HCC development. We showed that SET8 knockdown inhibited proliferation of HCC cells both in vitro and in vivo. In addition, SET8 knockdown inhibited migration and invasion of HCC cells. These results are in line with previous studies that highlighted the role of SET8 in progression and metastasis of a variety of tumours, such as papillary thyroid cancer, oesophageal squamous  www.nature.com/scientificreports www.nature.com/scientificreports/ cell carcinoma and renal cell carcinoma [26][27][28] . Consistent with these data, SET8 overexpression and UNC0379 treatment also proved that SET8 is an oncogene that favours HCC development. The fact that SET8 knockdown increased sensitivity of SMMC-7721 cells to docetaxel treatment implied the potential for SET8 to be used as a treatment target and biomarker for treatment efficiency. Docetaxel is a member of the taxane family that is known to disrupt microtubule formation during cell division. This drug has displayed antiproliferative activity through induction of HCC cell death in a previous study 29 . The potential prevention mechanism of docetaxel and its interaction with SET8 in HCC progression require further study in a larger sample size and in animal models.
A total of 406 genes were significantly altered in SET8 knockdown HCC cells, based on microarray analysis. Reactome FI analysis showed that genes altered in many biological processes referring to the cell cycle, cell proliferation, cell migration, cell adhesion, apoptosis, cytokine, angiogenesis, the Wnt signalling pathway,VEGF signalling pathway and p53 signalling pathway upon SET8 knockdown, which implied that these processes may be modified by SET8 alteration.
The Wnt signalling pathway is essential for a number of cellular functions including cell proliferation, migration and invasion 30 . SET8 knockdown inhibited proliferation, migration and invasion of renal carcinoma 786-O cells, potentially through Wnt/β-catenin signalling 28 . Our results also demonstrated that the Wnt signalling pathway may be responsible for modulating cellular functions upon SET8 knockdown. VEGF is a major driver of angiogenesis in carcinogenesis and cancer progression that correlates with vascular and portal vein invasion and influences HCC outcome 31 . In addition, the novel anti-VEGF humanised monoclonal antibody BD0801 significantly inhibited proliferation of SMMC-7721, and caused cell cycle arrest at the G1 phase 25 . Taken together, all these signalling pathways may modify the outcome of HCC upon SET8 knockdown. Functional analysis of candidate genes should be performed in further studies.
SET8 also catalyses monomethylation of H4K20me1 and of non-histone proteins, such as p53, TWIST, Wnt, and PCNA. Accumulating evidence has shown that SET8 is crucial for gene transcription regulation during tumour formation and progression 6,7,9 . It has been reported that SET8 modulates p53 activity through methylation at K382 and knockdown of SET8 resulted in p53 reduction in papillary thyroid cancer cells 26 . We did find that SET8 modulated p53 expression through methylation of K382 in HCC cells. SET8 may influence HCC development through methylation of p53 and subsequent downstream signal changes. Additionally, SET8 induced epithelial-mesenchymal transition (EMT) through interaction with TWIST, thereby enhancing cell invasion in breast cancer 12 . Moreover, SET8 interacts directly with the DNA replication factor PCNA and shows specific effect at the origins of replication 32 . Taken together, SET8 could play an oncogenic role by facilitating tumorigenesis.
In summary, our study suggests that SET8 may modify HCC development and progression by influencing proliferation, migration and invasion through modulation of a number of genes. Therefore, SET8 may be a potential new therapeutic target for HCC treatment.

Data availability
All data generated or analysis during this study are included in this published article (and its Supplementary Information files).