SET7/9 promotes hepatocellular carcinoma progression through regulation of E2F1

Abstract

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide. Histone‑lysine N‑methyltransferase SET7/9 is a protein lysine monomethylase that methylates histone H3K4 as well as various non‑histone proteins. Deregulation of SET7/9 is frequently detected in human cancers. However, the role of SET7/9 in HCC development remains unclear. In the present study, upregulation of SET7/9 and E2F transcription factor 1 (E2F1) expression was detected in 68 samples of HCC tissues compared with these levels noted in the paired healthy liver samples. The expression levels of SET7/9 and E2F1 were significantly correlated with pathological stage and tumor size. Subcellular fractionation and co‑immunoprecipitation analyses revealed protein‑protein interaction between SET7/9 and E2F1 in the cytoplasm of HCC cells. Silencing of SET7/9, as well as treatment with 5'‑deoxy‑5'‑methylthioadenosine (MTA), a protein methylation inhibitor, led to reduced E2F1 protein abundance in HCC cells. Using Cell Counting Kit‑8 (CCK‑8) assay, Transwell migration assay and wound healing assay, significantly decreased cell proliferation, migration and invasion were observed in cells exhibiting downregulation of SET7/9 and E2F1 expression, as well as in wild‑type HCC cells treated with MTA. Furthermore, SET7/9 downregulation and MTA treatment resulted in reduced expression of downstream targets of E2F1, including cyclin A2, cyclin E1 and CDK2. In conclusion, the present study revealed an oncogenic function of SET7/9 in HCC and demonstrated that SET7/9 may be responsible for alterations in the proliferative ability, aggressiveness and invasive/metastatic potential of HCC cells through post‑translational regulation of E2F1.

Figure 1.

Expression of SET7/9 in clinical HCC samples and cell lines. (A) Immunostaining of SET7/9 protein in HCC tissues and the surrounding non-cancerous tissues. (a) Negative expression (0) in healthy liver tissue. (b) Weak expression (1+) in HCC tissues. (c) Moderate expression (2+) in HCC tissues. (d) Strong expression (3+) in HCC tissues. (B) Staining scores of SET7/9 protein expression in HCC tissue samples and surrounding noncancerous tissues. The staining score ranged from 0 to 3, with 0 for no staining, 1 for weak staining, 2 for moderate staining, and 3 for strong staining. *P<0.05. (C) Expression of SET7/9 and E2F1 in three human HCC cell lines Huh7, Hep3B, and SK-HEP-1, and the human healthy liver cell line LO2. HCC, hepatocellular carcinoma.

Figure 2.

Expression of E2F1 in clinical HCC samples. (A) Immunostaining of E2F1 protein in HCC tissues and the surrounding non-cancerous tissues. (a) Negative expression (0) in healthy liver tissues. (b) Weak expression (1+) in HCC tissues. (c) Moderate expression (2+) in HCC tissues. (d) Strong expression (3+) in HCC tissues. (B) Staining scores of E2F1 protein expression in HCC tissue samples and surrounding noncancerous tissues. The staining score ranged from 0 to 3, with 0 for no staining, 1 for weak staining, 2 for moderate staining, and 3 for strong staining. *P<0.05. (C) Staining scores of E2F1 protein expression in HCC tissue samples with T1, T2, T3, and T4 stage and surrounding noncancerous tissues. (D) Staining scores of E2F1 protein expression in HCC tissue samples with N0 and N1 stage and surrounding noncancerous tissues. (E) Staining scores of E2F1 protein expression in HCC tissue samples with M0 and M1 stage and surrounding noncancerous tissues. T, tumor size; N, regional lymph node metastasis; M, distant metastatsis; HCC, hepatocellular carcinoma.

Figure 3.

SET7/9 directly interacts with E2F1 and regulates E2F1 abundance through post-translational methylation in HCC cells. (A) E2F1 expression at the protein level was downregulated in SET7/9-silenced cells and MTA-treated cells as compared with the control cells. (B) E2F1 showed similar mRNA expression levels in SET7/9-silenced cells, MTA-treated cells, and the control cells. (C) SET7/9 expression at the protein level did not show significant change after E2F1 downregulation and MTA treatment as compared with the control cells. (D) SET7/9 showed similar mRNA expression levels in E2F1-silenced cells, MTA-treated cells, and the control cells. (E) Co-immunoprecipitation of total cell and subcellular fractions for SET7/9 and E2F1 in the HCC cell line Huh7. Lysates were immunoprecipitated with SET7/9 antibody or control IgG and detected with E2F1 antibody on a western blotting, then immunoprecipitated with E2F1 antibody or control IgG and detected with SET7/9 antibody on a western blotting. Each error bar represents the mean ± SD of three replicate samples. HCC, hepatocellular carcinoma.

Figure 4.

Silencing of SET7/9 leads to changes in cellular behavior of human HCC cells. (A) SET7/9 downregulation and MTA treatment significantly reduced cell proliferation as determined by cell proliferation assay. (B) SET7/9-silenced HCC cells as well as MTA-treated cells showed decreased migration and invasion through extracellular matrix (ECM) as indicated by Transwell migration assay. Representative images (left) and quantification (right) are shown. The number of cells that migrated through the ECM after 24 h was counted in five randomly selected (×200) microscopic fields. (C) SET7/9 downregulation and MTA treatment led to reduced cell invasive ability as revealed by wound healing assay. Images were captured at 0 and 24 h. At 24 h, the extent of wound closure was 31.33% (±3.20%) for SET7/9-silenced cells, 33.00% (±5.06%) for MTA-treated cells, but was 79.50% (±3.73%) for the control cells (NC) transduced with scrambled-shRNA vectors. Each error bar represents the mean ± SD of three replicate samples. *P<0.05. HCC, hepatocellular carcinoma.

Figure 5.

Silencing of E2F1 leads to changes in cellular behavior of human HCC cells. (A) E2F1 downregulation and MTA treatment significantly reduced cell proliferation as determined by cell proliferation assay. (B) E2F1-silenced HCC cells as well as MTA-treated cells showed decreased migration and invasion through extracellular matrix (ECM) as indicated by Transwell migration assay. Representative images (left) and quantification (right) are shown. The number of cells migrated through the ECM after 24 h was counted in five randomly selected (×200) microscopic fields. (C) E2F1 downregulation and MTA treatment led to reduced cell invasive ability as revealed by wound healing assay. At 24 h, the extent of wound closure was 33.17% (±2.79%) for E2F1-silenced cells, 37.67% (±1.86%) for MTA-treated cells, but was 95.00% (±2.61%) for the control cells (NC) transduced with scrambled-shRNA vectors. Each error bar represents the mean ± SD of three replicate samples. *P<0.05. HCC, hepatocellular carcinoma.

Figure 6.

Silencing of SET7/9 leads to changes in the expression of E2F1 downstream targets. Downregulation of E2F1, cyclin E1, cyclin A2, and CDK2 was observed in both the SET7/9-silenced and the MTA-treated cells.

Figure 7.

SET7/9 expression profiling from public databases. (A-F) Box plots from the TCGA database showing the differences in the expression level of SET7/9 in normal and tumor tissue from various types of cancer. (A) Colon adenocarcinoma (COAD), (B) head and neck squamous cell carcinoma (HNSC), (C) kidney renal clear cell carcinoma (KIRC), (D) kidney renal papillary cell carcinoma (KIRP), (E) pancreatic adenocarcinoma (PAAD), (F) uveal melanoma (UVM). (G) Summary of SET7/9 expression profiling in different cancer types in the ONCOMINE database. The expression differences in all box plots are significant with a P-value <0.05.